RESUMEN
Annexin A6 is a multicompetent, multifunctional protein involved in several biological processes within and outside of the cell. Whereas HeLa cells express annexin A6 only as a 68/67-kDa doublet, indicating alternative splicing (Smith PD et al. (1994) Proc Natl Acad Sci USA 91, 2713-2717), the GMO2784 human fibroblast cell line expresses two additional isoforms at 64 and 58kDa. In both cell lines, annexin A6 is located intracellularly and on the plasma membrane. In vitro eukaryotic protein synthesis of pIRESneoAnxA6 cDNA and pIRESneoAnxA6/Met1- or Met33- using a reticulocyte lysate coupled transcription/translation system revealed that this gene contains two translation start codons, Met1 and Met33. Immunoprecipitation of the products obtained from the transcription/translation system using various anti-annexin A6 antibodies confirmed the presence of several isoforms and suggested that this protein might be present in different configurations.
Asunto(s)
Empalme Alternativo/genética , Anexina A6/genética , Iniciación de la Cadena Peptídica Traduccional , Isoformas de Proteínas/genética , Anexina A6/biosíntesis , Secuencia de Bases , Línea Celular , Membrana Celular , Codón Iniciador/genética , ADN Complementario , Fibroblastos , Regulación de la Expresión Génica/genética , Humanos , Isoformas de Proteínas/biosíntesisRESUMEN
In human cells, HCS catalyzes the biotinylation of biotin-dependent carboxylases and mediates the transcriptional control of genes involved in biotin metabolism through the activation of a cGMP-dependent signal transduction pathway. HCS also targets to the cell nucleus in association with lamin-B suggesting additional gene regulatory functions. Studies from our laboratory in Drosophila melanogaster showed that nuclear HCS is associated with heterochromatin bands enriched with the transcriptionally repressive mark histone 3 trimethylated at lysine 9. Further, HCS was shown to be recruited to the core promoter of the transcriptionally inactive hsp70 gene suggesting that it may participate in the repression of gene expression, although the mechanism involved remained elusive. In this work, we expressed HCS as a fusion protein with the DNA-binding domain of GAL4 to evaluate its effect on the transcription of a luciferase reporter gene. We show that HCS possesses transcriptional repressor activity in HepG2 cells. The transcriptional function of HCS was shown by in vitro pull down and in vivo co-immunoprecipitation assays to depend on its interaction with the histone deacetylases HDAC1, HDAC2 and HDAC7. We show further that HCS interaction with HDACs and its function in transcriptional repression is not affected by mutations impairing its biotin-ligase activity. We propose that nuclear HCS mediates events of transcriptional repression through a biotin-independent mechanism that involves its interaction with chromatin-modifying protein complexes that include histone deacetylases.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Histona Desacetilasas/genética , Biotina/metabolismo , Ligasas de Carbono-Nitrógeno/genética , Cromatina , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Células Hep G2 , Heterocromatina/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/metabolismo , Histona Desacetilasas/metabolismo , Humanos , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Intracellular pathogens, such as Mycobacterium tuberculosis, reside in the phagosomes of macrophages where antigenic processing is initiated. Mycobacterial antigen-MHC class II complexes are formed within the phagosome and are then trafficked to the cell surface. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) influence the outcome of M. tuberculosis infection; however, the role of these cytokines with regard to the formation of M. tuberculosis peptide-MHC-II complexes remains unknown. We analysed the kinetics and subcellular localization of M. tuberculosis peptide-MHC-II complexes in M. tuberculosis-infected human monocyte-derived macrophages (MDMs) using autologous M. tuberculosis-specific CD4(+) T cells. The MDMs were pre-treated with either IFN-γ or IL-10 and infected with M. tuberculosis. Cells were mechanically homogenized, separated on Percoll density gradients and manually fractionated. The fractions were incubated with autologous M. tuberculosis -specific CD4(+) T cells. Our results demonstrated that in MDMs pre-treated with IFN-γ, M. tuberculosis peptide-MHC-II complexes were detected early mainly in the phagosomal fractions, whereas in the absence of IFN-γ, the complexes were detected in the endosomal fractions. In MDMs pre-treated with IL-10, the M. tuberculosis peptide-MHC-II complexes were retained in the endosomal fractions, and these complexes were not detected in the plasma membrane fractions. The results of immunofluorescence microscopy demonstrated the presence of Ag85B associated with HLA-DR at the cell surface only in the IFN-γ-treated MDMs, suggesting that IFN-γ may accelerate M. tuberculosis antigen processing and presentation at the cell membrane, whereas IL-10 favours the trafficking of Ag85B to vesicles that do not contain LAMP-1. Therefore, IFN-γ and IL-10 play a role in the formation and trafficking of M. tuberculosis peptide-MHC-II complexes.
Asunto(s)
Antígenos Bacterianos/inmunología , Interferón gamma/inmunología , Interleucina-10/inmunología , Fagosomas/inmunología , Línea Celular , Humanos , Mycobacterium tuberculosis/inmunologíaRESUMEN
Endocytosis and transport of bovine liver ß-glucuronidase to lysosomes in human fibroblasts are mediated by two receptors: the well-characterized cation-independent mannose 6-phosphate receptor (IGF-II/Man6PR) and an IGF-II/Man6PR-independent receptor, which recognizes a Ser-Trp*-Ser sequence present on the ligand. The latter receptor was detergent extracted from bovine liver membranes and purified. LC/ESI-MS/MS analysis revealed that this endocytic receptor was annexin VI (AnxA6). Several approaches were used to confirm this finding. First, the binding of bovine ß-glucuronidase to the purified receptor from bovine liver membranes and His-tagged recombinant human AnxA6 protein was confirmed using ligand-blotting assays. Second, western blot analysis using antibodies raised against IGF-II/Man6PR-independent receptor as well as commercial antibodies against AnxA6 confirmed that the receptor and AnxA6 were indeed the same protein. Third, double immunofluorescence experiments in human fibroblasts confirmed a complete colocalization of the bovine ß-glucuronidase and the AnxA6 receptor on the plasma membrane. Lastly, two cell lines were stably transfected with a plasmid containing the cDNA for human AnxA6. In both transfected cell lines, an increase in cell surface AnxA6 and in mannose 6-phosphate-independent endocytosis of bovine ß-glucuronidase was detected. These results indicate that AnxA6 is a novel receptor that mediates the endocytosis of the bovine ß-glucuronidase.
Asunto(s)
Anexina A6/fisiología , Endocitosis/fisiología , Glucuronidasa/metabolismo , Receptor IGF Tipo 2/fisiología , Receptores de Superficie Celular/fisiología , Animales , Anexina A6/análisis , Anexina A6/aislamiento & purificación , Anticuerpos/inmunología , Anticuerpos/farmacología , Bovinos , Línea Celular Tumoral , Endocitosis/efectos de los fármacos , Células Epiteliales/fisiología , Fibroblastos/efectos de los fármacos , Fibroblastos/fisiología , Humanos , Células L , Hígado/química , Hígado/enzimología , Manosafosfatos/farmacología , Espectrometría de Masas , Ratones , Unión Proteica/fisiología , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Transfección , Vesículas Transportadoras/metabolismoRESUMEN
Extracellular and intraneuronal formation of amyloid-beta aggregates have been demonstrated to be involved in the pathogenesis of Alzheimer's disease. However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of targets have deleterious effects on cellular functions. In the present study we have shown for the first time that amyloid-beta 1-42 bound to a peptide comprising the microtubule binding domain of the heavy chain of microtubule-associated protein 1B by the screening of a human brain cDNA library expressed on M13 phage. This interaction may explain, in part, the loss of neuronal cytoskeletal integrity, impairment of microtubule-dependent transport and synaptic dysfunction observed previously in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Encéfalo/fisiopatología , Línea Celular Tumoral , Humanos , Microtúbulos/metabolismo , Péptidos/metabolismo , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Transporte de Proteínas/fisiología , Transmisión Sináptica/fisiologíaRESUMEN
Human mitochondrial methylmalonyl-CoA mutase (hMCM) is an isomerase that converts methylmalonyl-CoA to succinyl-CoA, a crucial step for the incorporation of some compounds derived from the diet into the central metabolism. hMCM employs highly reactive radicals from its cofactor (adenosylcobalamin, AdoCbl) to perform its reaction. Our previous work demonstrated that hMCM loses activity during catalysis and that the interaction with human MMAA (hMMAA), a GTPase protein, avoided this loss or restored hMCM activity. Even so, the mechanism by which hMMAA exerted these chaperone functions has not been described. In this work report that the formation and accumulation of OH2Cbl, the oxidized form of the AdoCbl cofactor formed during catalysis, is the cause of hMCM inactivation. Additionally, we demonstrate that the complex formation of hMCM/hMMAA decreases the rate of oxidized cofactor formation, protecting the hMCM enzyme. Moreover, an inactive model of hMCM was used to demonstrate that hMMAA is able to remove the damaged cofactor through GTP hydrolysis. Additionally, a modification in the kinetic parameters of hMCM in presence of hMMAA was observed, and for the first time, the in vivo localization of hMMAA and its colocalization with hMCM in human fibroblasts mitochondria were demonstrated.
Asunto(s)
Coenzimas/metabolismo , Metilmalonil-CoA Mutasa/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Fibroblastos/metabolismo , Humanos , Mitocondrias/metabolismo , Oxidación-Reducción , Transporte de ProteínasRESUMEN
The distribution of the cation-independent mannose 6-phosphate and 78 kDa receptors was studied in postnuclear subcellular fractions from two rat liver cell lines. ELISA assays revealed that the mannose 6-phosphate receptor is enriched in the light buoyant Percoll fractions that contain Golgi structures and early endosomes. Most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient and smaller amounts in the endosomal fractions. The high-density compartment is denser than lysosomes, contains LAMP2 but not LIMPII or acid hydrolases, and is not disrupted with glycyl-l-phenylalanine 2-naphthylamide, a substrate for cathepsin C that selectively disrupts lysosomes. Immunofluorescence microscopy studies indicate no colocalization of the 78 kDa receptor with the mannose 6-phosphate receptor or LIMPII. Mannose 6-phosphate-independent endocytosed beta-glucuronidase was found in the lysosomal, the early and late endosomal fractions. These fractions were immunoadsorbed in columns containing antibodies against the 78 kDa receptor. Only the endocytosed beta-glucuronidase present in the early and late endosomal fractions is associated to immunoadsorbed vesicles. In these vesicles, LAMP2 was detected but no LIMPII or the mannose 6-phosphate receptor. Results obtained suggest that the 78 kDa receptor is found along the endocytic pathway, but in vesicles different from the cation-independent mannose 6-phosphate receptor.
Asunto(s)
Hepatocitos/metabolismo , Hígado/metabolismo , Lisosomas/enzimología , Manosafosfatos/metabolismo , Receptor IGF Tipo 2/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Fraccionamiento Celular , Línea Celular , Centrifugación por Gradiente de Densidad , Técnica del Anticuerpo Fluorescente Indirecta , Hepatocitos/ultraestructura , Hígado/ultraestructura , Peso Molecular , Povidona , Ratas , Receptor IGF Tipo 2/aislamiento & purificación , Dióxido de SilicioRESUMEN
Recent work has shown that the cation-independent mannose 6-phosphate and the 78 kDa receptors for lysosomal enzyme targeting are located in different cell compartments. While the mannose 6-phosphate receptor is enriched in the Percoll fractions that contain Golgi apparatus, most of the 78 kDa receptor is localized in a heavy fraction at the bottom of the Percoll gradient. This report presents the biosynthetic transport of the 78 kDa receptor. Newly synthesized 78 kDa receptor was transported to Golgi from endoplasmic reticulum with a half life of 5 min. From the Golgi apparatus, the receptor takes two routes; about 15-25% is transported to the plasma membrane, and the rest migrates to late endosomes, subsequently to prelysosomes and finally to the dense vesicles. The 78 kDa receptor starts appearing at the dense vesicles 120 min after biosynthesis and reaches a maximum of 40-50% of the total receptor. Treatment of cells with NH4Cl causes depletion of the receptor from the dense vesicles and prelysosomes and corresponding augmentation in endosomes and plasma membrane. These results suggest that the 78 kDa receptor cycles between compartments and that the dense vesicles seem to represent the most distal compartment in the biosynthetic pathway of this receptor.
Asunto(s)
Retículo Endoplásmico/metabolismo , Lisosomas/enzimología , Manosafosfatos/metabolismo , Receptor IGF Tipo 2/biosíntesis , Receptor IGF Tipo 2/metabolismo , Cloruro de Amonio , Línea Celular , Centrifugación por Gradiente de Densidad , Retículo Endoplásmico/enzimología , Hepatocitos/enzimología , Hepatocitos/metabolismo , Humanos , Líquido Intracelular/metabolismo , Cinética , Lisosomas/metabolismo , Peso Molecular , Octoxinol , Povidona , Transporte de Proteínas/fisiología , Receptor IGF Tipo 2/química , Dióxido de SilicioRESUMEN
In human cells, biotin is essential to maintain metabolic homeostasis and as regulator of gene expression. The enzyme holocarboxylase synthetase (HCS) transforms biotin into its active form 5'-biotinyl-AMP and this compound is used to biotinylate five biotin-dependent carboxylases or to activate a soluble guanylate cyclase (sGC) and a cGMP-dependent protein kinase (PKG). The HCS-sGC-PKG pathway is responsible for maintaining the mRNA levels of enzymes involved in biotin utilization including HCS, carboxylases, and a biotin carrier known as sodium-dependent multivitamin transporter (SMVT). To understand the role of SMVT in the control of biotin utilization, we have studied the effect of biotin availability on SMVT protein and mRNA expression levels in HepG2 cells by Western blot analysis and rtPCR, respectively; and their functional impact on the rate of [3H]biotin uptake in human cells. Our results showed that human HepG2 cells grown in a biotin-deficient medium have a lower rate of biotin uptake than normal cells. The impairment in biotin uptake is associated with a reduction in the amount of both SMVT protein mass and mRNA levels. Transfection of HepG2 cells with a vector containing a luciferase reporter gene under the control of the rat SMVT promoter demonstrated that its transcriptional activity is regulated by biotin availability through activation of the HCS-sGC-PKG pathway. Our results support the proposed role of SMVT in the altruistic regulation of biotin utilization in liver cells that has been associated with sparing biotin depletion of the brain.