RESUMEN
The P2X7 receptor (P2X7R) is an ATP-gated cationic channel expressed by hematopoietic, epithelial, and neuronal cells. Prolonged ATP exposure leads to the formation of a nonselective pore, which can result in cell death. We show that P2X7R is associated with detergent-resistant membranes (DRMs) in both transfected human embryonic kidney (HEK) cells and primary macrophages independently from ATP binding. The DRM association requires the posttranslational modification of P2X7R by palmitic acid. Treatment of cells with the palmitic acid analog 2-bromopalmitate as well as mutations of cysteine to alanine residues abolished P2X7R palmitoylation. Substitution of the 17 intracellular cysteines of P2X7R revealed that 4 regions of the carboxyl terminus domain are involved in palmitoylation. Palmitoylation-defective P2X7R mutants showed a dramatic decrease in cell surface expression because of their retention in the endoplasmic reticulum and proteolytic degradation. Taken together, our data demonstrate that P2X7R palmitoylation plays a critical role in its association with the lipid microdomains of the plasma membrane and in the regulation of its half-life.
Asunto(s)
Adenosina Trifosfato/metabolismo , Activación del Canal Iónico/fisiología , Lipoilación/fisiología , Microdominios de Membrana/fisiología , Receptores Purinérgicos P2/metabolismo , Secuencia de Aminoácidos , Línea Celular , Membrana Celular/fisiología , Semivida , Humanos , Macrófagos , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7RESUMEN
Presenilin 1 (PS1) mutations account for the majority of early-onset dominant cases of familial Alzheimer's disease. Presenilins (PSs) are located in many intra-cellular compartments such as the endoplasmic reticulum, Golgi apparatus, nuclear region and vesicular structures. These proteins include from seven to nine putative transmembrane domains, with the N- and C-terminal ends and a large hydrophilic loop orientated towards the cytoplasm. We report an interaction between the human PS1 or PS2 hydrophilic loop and Rab11, a small GTPase belonging to the Ras-related superfamily. Interaction domains were mapped to codons 374-400 for PS1 and to codons 106-179 for Rab11, a region including the fourth GTP-binding domain. Considering the implication of Rab proteins in vesicular transport pathways, the PS-Rab11 inter-action suggests that PSs might be involved in amyloid precursor protein vesicular routing.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al GTP rab , Secuencia de Aminoácidos , Animales , Sitios de Unión , Transporte Biológico/fisiología , Células COS , Humanos , Datos de Secuencia Molecular , Presenilina-1 , Conformación Proteica , Saccharomyces cerevisiae/metabolismoRESUMEN
The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated'). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic gamma-tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.
Asunto(s)
Proteínas Portadoras/metabolismo , Centrosoma/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas de Unión al GTP rab , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Activación Enzimática , Proteínas Fúngicas/genética , Proteínas Activadoras de GTPasa , Células HeLa , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Activadoras de ras GTPasaRESUMEN
We previously demonstrated that, in hepatocytes in primary culture, the role of insulin on induction of L-type pyruvate kinase (L-PK) gene expression was mainly to induce glucokinase synthesis, needed for glucose phosphorylation to glucose 6-phosphate. However, we show here that when hepatocytes have been isolated from rats starved for 72 h, glucose and constitutive glucokinase expression was not sufficient to fully stimulate the L-PK promoter, low insulin concentrations being still required. In addition, activation remains sensitive to cAMP inhibition, but cannot be reproduced in the absence of insulin by a competitive cAMP antagonist. We propose that both insulin and cAMP act on expression of the L-PK gene at, at least, two levels: positive and negative regulation of glucokinase gene expression, and more downstream levels.
Asunto(s)
AMP Cíclico/metabolismo , Insulina/metabolismo , Regiones Promotoras Genéticas , Piruvato Quinasa/genética , Transcripción Genética , Animales , Células Cultivadas , AMP Cíclico/farmacología , Regulación de la Expresión Génica , Glucoquinasa/metabolismo , Glucosa/metabolismo , Insulina/farmacología , Hígado/citología , Masculino , Ratas , Ratas Sprague-Dawley , Activación TranscripcionalAsunto(s)
Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Hígado/enzimología , Regiones Promotoras Genéticas , Piruvato Quinasa/biosíntesis , Animales , Línea Celular , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/biosíntesis , Inducción Enzimática , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Insulina/farmacología , Neoplasias Hepáticas Experimentales , Regiones Promotoras Genéticas/efectos de los fármacos , Piruvato Quinasa/genética , Proteínas Recombinantes de Fusión/biosíntesis , Eliminación de Secuencia , Transfección , Xilitol/farmacologíaRESUMEN
Glucose catabolism induces the expression of the L-type pyruvate kinase (L-PK) gene through the glucose response element (GIRE). The metabolic pathway used by glucose after its phosphorylation to glucose 6-phosphate by glucokinase to induce L-PK gene expression in hepatocytes remains unknown. The sugar alcohol xylitol is metabolized to xylulose 5-phosphate, an intermediate of the nonoxidative branch of the pentose phosphate pathway. In this study, we demonstrated that xylitol at low concentration (O.5 mM) induced the expression of the L-PK/CAT construct in glucose-responsive mhAT3F hepatoma cells at the same level as 20 mM glucose, while it did not affect intracellular concentration of glucose 6-phosphate significantly. The effect of xylitol on the induction of the L-PK gene expression was noncumulative with that of glucose since 20 mM glucose plus 5 mM xylitol induced the expression of the L-PK/CAT construct similarly to 20 mM glucose alone. In hepatocytes in primary culture, 5 mM xylitol induced accumulation of the L-PK mRNA even in the absence of insulin. Furthermore, the response to xylitol as well as glucose required the presence of a functional GIRE. It can be assumed from these results that glucose induces the expression of the L-PK gene through the nonoxidative branch of the pentose phosphate pathway. The effect of xylitol at low concentration suggests that the glucose signal to the transcriptional machinery is mediated by xylulose 5-phosphate.
Asunto(s)
Glucosa/farmacología , Isoenzimas/biosíntesis , Hígado/metabolismo , Vía de Pentosa Fosfato , Piruvato Quinasa/biosíntesis , Transducción de Señal , Transcripción Genética , Xilitol/farmacología , Animales , Northern Blotting , Línea Celular , Células Cultivadas , Cloranfenicol O-Acetiltransferasa/biosíntesis , Inducción Enzimática , Expresión Génica , Glucoquinasa/metabolismo , Glucosa-6-Fosfato , Glucofosfatos/análisis , Glucofosfatos/metabolismo , Cinética , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Transgénicos , Vía de Pentosa Fosfato/efectos de los fármacos , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway mainly expressed in the liver. Rat liver contains a regulatory protein that inhibits glucokinase (GK) activity. The effect of this protein is greatly reinforced by the fructose 6-phosphate and antagonized by the fructose 1-phosphate (Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184). In hepatocytes, fructose in low concentrations is phosphorylated into fructose 1-phosphate, and therefore is able to active GK in the absence of insulin via the regulatory protein in the liver. In primary culture of rat hepatocytes, 0.2 mM fructose in the presence of 20 or 40 mM glucose stimulated the activity of the L-PK gene promoter fused with the chloramphenicol acetyltransferase reporter gene, regardless of the addition of insulin, through the glucose/insulin response element. A constitutive GK expression vector co-transfected with the L-PK/chloramphenicol acetyltransferase construct is also able to confer an insulin-independent glucose responsiveness in hepatocytes. Thus, the insulin effect on glucose-dependent activation of the L-PK promoter is, under these experimental conditions, to permit glucose phosphorylation through the stimulation of the GK synthesis. In the presence of glucose, the L-PK promoter can also be activated by a post-translational GK activation, mediated by a low concentration of fructose acting via the regulatory protein of glucokinase.
Asunto(s)
Fructosa/fisiología , Glucosa/fisiología , Insulina/fisiología , Hígado/enzimología , Regiones Promotoras Genéticas , Piruvato Quinasa/genética , Animales , Células Cultivadas , Regulación Enzimológica de la Expresión Génica , Vectores Genéticos , Glucoquinasa/antagonistas & inhibidores , Glucoquinasa/genética , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.
Asunto(s)
Glucosa/farmacología , Fosfoproteínas , Piruvato Quinasa/genética , Animales , Secuencia de Bases , Sitios de Unión , ADN/genética , Proteínas de Unión al ADN/metabolismo , Factor Nuclear 4 del Hepatocito , Insulina/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Piruvato Quinasa/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/metabolismo , Factores Estimuladores hacia 5'RESUMEN
The functional role of the different sites binding transcriptional factors on the tissue-specific, glucose-responsive promoter of the L type pyruvate kinase gene (L-PK) has been investigated in transgenic mice. These sites are able to bind, from 3' to 5', HNF1, NF1, HNF4, and MLTF/USF, respectively. We have compared the level of chloramphenicol acetyltransferase reporter transgene expression when driven by a L-PK promoter fragment of either -96 base pairs (bp) (containing only the HNF1 binding site) or -150 bp (lacking the MLTF/USF binding site) or driven by a -183-bp L-PK promoter fragment with or without the NF1 binding site. Our results demonstrate that: 1) HNF1 alone is not sufficient to promote an efficient L-PK gene transcription in vivo; 2) with only binding sites for HNF1, NF1, and HNF4, though the tissue-specific pattern of expression is respected, the level of the gene transcription is low and the hormonal control is lost; 3) the MLTF/USF binding site is the target of the hormonal control, required for both positive response to carbohydrates and negative response to glucagon; 4) the role of NF1 in the promoter activity could be to negatively modulate the L-PK gene expression in the different tissues, without interfering with the glucose and hormone responsiveness.
Asunto(s)
Encéfalo/metabolismo , Cloranfenicol O-Acetiltransferasa/genética , Glucosa/farmacología , Insulina/farmacología , Hígado/metabolismo , Regiones Promotoras Genéticas , Piruvato Quinasa/genética , Factores de Transcripción/metabolismo , Animales , Northern Blotting , Cloranfenicol O-Acetiltransferasa/biosíntesis , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/efectos de los fármacos , Transcripción GenéticaRESUMEN
L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway specifically expressed in the liver and, to a lesser degree, in the small intestine and kidney. One important characteristic of L-PK gene expression is its strong activation by glucose and insulin and its complete inhibition by fasting or glucagon treatment. Having previously established that the entire rat L-PK gene plus 3.2 kbp of 5'-flanking region functions in mice in a tissue-specific and hormonally regulated manner, various deletions of these 3.2 kbp of 5'-flanking regions were tested in transgenic animals to map the cis-acting elements involved in transcriptional gene regulation. Our experiments indicate that the proximal region between -183 and +11 confers tissue specificity and contains all the information necessary for dietary and hormonal control of L-PK gene expression in vivo. We found, however, that the transcriptional activity generated by this proximal promoter fragment can be modulated by distal sequences in a tissue-specific manner. (i) Sequences between bp -183 and -392 seem to play a dual role in the liver and small intestine; they induce L-PK expression in the liver but repress it in the small intestine. (ii) Sequences from bp -392 up to -1170 do not seem to have any additional effect on promoter activity. (iii) Between bp -1170 and -2080, we found a putative extinguisher whose transcriptional inhibitory effect is much more marked in the small intestine than in the liver. (iv) Finally, between bp -2080 and -3200, we identified an activating sequence required for full expression of the gene in the liver.