RESUMEN
Interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) by the UDP-Glc 4´-epimerase intimately connects the biosynthesis of these two nucleotide sugars. Their de novo biosynthesis involves transformation of glucose-6-phosphate into glucose-1-phosphate by the phosphoglucomutase and subsequent activation into UDP-Glc by the specific UDP-Glc pyrophosphorylase (UGP). Besides UGP, Leishmania parasites express an uncommon UDP-sugar pyrophosphorylase (USP) able to activate both galactose-1-phosphate and glucose-1-phosphate in vitro. Targeted gene deletion of UGP alone was previously shown to principally affect expression of lipophosphoglycan, resulting in a reduced virulence. Since our attempts to delete both UGP and USP failed, deletion of UGP was combined with conditional destabilisation of USP to control the biosynthesis of UDP-Glc and UDP-Gal. Stabilisation of the enzyme produced by a single USP allele was sufficient to maintain the steady-state pools of these two nucleotide sugars and preserve almost normal glycoinositolphospholipids galactosylation, but at the apparent expense of lipophosphoglycan biosynthesis. However, under destabilising conditions, the absence of both UGP and USP resulted in depletion of UDP-Glc and UDP-Gal and led to growth cessation and cell death, suggesting that either or both of these metabolites is/are essential.
Asunto(s)
Leishmania major/crecimiento & desarrollo , Leishmania major/metabolismo , Uridina Difosfato Galactosa/deficiencia , Uridina Difosfato Glucosa/deficiencia , Eliminación de Gen , Regulación de la Expresión Génica , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato Glucosa/metabolismoRESUMEN
The effects of glucose starvation on glycogen synthase (GS) activity and protein expression were investigated. Fibroblasts were cultured in medium supplemented with either glucose or pyruvate. Pyruvate-cultured cells exhibited UDP-glucose contents that amounted to approximately 10% of those in cells cultured with glucose. GS activity, protein and mRNA amounts in pyruvate-cultured cells were decreased to approximately 35, 60, and 60%, respectively, of values in glucose-cultured cells. Incubation of extracts from glucose-cultured cells with radioactive UDP-glucose resulted in substantial binding of ligand to immunoprecipitated GS. However, binding in immunoprecipitates from pyruvate-cultured cells was decreased to approximately 25% of values in glucose-cultured cells. These data indicate that glucose starvation and the subsequent depletion of UDP-glucose result in: (1) inactivation of GS, owing to a decrease in its ability to bind UDP-glucose, and (2) decreased amount of GS protein, owing to a decrease in the levels of GS mRNA.
Asunto(s)
Fibroblastos/metabolismo , Glucosa/deficiencia , Glucógeno Sintasa/metabolismo , Uridina Difosfato Glucosa/deficiencia , Animales , Células Cultivadas , Cricetinae , Cricetulus , Activación Enzimática , Inhibidores Enzimáticos , Pulmón/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Inanición/metabolismoRESUMEN
A low level of UDP-Glc occurs in cells exposed to hypoxia or glucose starvation. This work reveals that a 65% reduction in the cellular UDP-Glc level causes up-regulation of the mitochondrial chaperone GRP75 and the endoplasmic reticulum (ER) resident chaperones GRP58, ERp72, GRP78, GRP94, GRP170, and calreticulin. Conditions that cause misfolding of proteins within the ER activate the transcription factors ATF6alpha/beta and induce translation of the transcription factors XBP-1/TREB5 and ATF4/CREB2. These transcription factors induce the overexpression of ER chaperones and CHOP/GADD153. However, the 65% decrease in the cellular UDP-Glc level does not cause activation of ATF6alpha, splicing of XBP-1/TREB5, induction of ATF4/CREB2, or expression of CHOP/GADD153. The activity of the promoters of the ER chaperones is increased in UDP-Glc-deficient cells, but the activity of the CHOP/GADD153 promoter is not affected, in comparison with their respective activities in cells having compensated for the UDP-Glc deficiency. The results demonstrate that the unfolded protein response remains functionally intact in cells with a 65% decrease in the cellular UDP-Glc level and provide evidence that this decrease is a stress signal in mammalian cells, which triggers the coordinate overexpression of mitochondrial and ER chaperones, independently of the ER stress elements.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glucosa/metabolismo , Oxígeno/metabolismo , Uridina Difosfato Glucosa/deficiencia , Factor de Transcripción Activador 6 , Adenosina Trifosfato/química , Alelos , Empalme Alternativo , Animales , Western Blotting , Proteínas Potenciadoras de Unión a CCAAT/genética , Calreticulina/biosíntesis , Proteínas Portadoras/biosíntesis , Línea Celular , Supervivencia Celular , Cricetinae , Citoplasma/metabolismo , Proteínas de Unión al ADN/metabolismo , Electroforesis en Gel Bidimensional , Chaperón BiP del Retículo Endoplásmico , Fibroblastos/metabolismo , Genes Dominantes , Genes Reporteros , Glicoproteínas/biosíntesis , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/biosíntesis , Hipoxia , Isomerasas/biosíntesis , Luciferasas/metabolismo , Espectroscopía de Resonancia Magnética , Glicoproteínas de Membrana/biosíntesis , Proteínas de la Membrana/biosíntesis , Mitocondrias/metabolismo , Chaperonas Moleculares/biosíntesis , Mutación , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , Transducción de Señal , Factor de Transcripción CHOP , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Tunicamicina/farmacología , Regulación hacia ArribaRESUMEN
Previously we reported that stable transfection of human UDP-glucose pyrophosphorylase (hUGP2) rescued galactose-1-phosphate uridyltransferase (GALT)-deficient yeast from "galactose toxicity." Here we test in human cell lines the hypothesis that galactose toxicity was caused by excess accumulation of galactose-1-phosphate (Gal-1-P), inhibition of hUGP2, and UDP-hexose deficiency. We found that SV40-transformed fibroblasts derived from a galactosemic patient accumulated Gal-1-P from 1.2+/-0.4 to 5.2+/-0.5 mM and stopped growing when transferred from 0.1% glucose to 0.1% galactose. Control fibroblasts accumulated little Gal-1-P and continued to grow. The GALT-deficient cells had 157+/-10 micromoles UDP-glucose/100 g protein and 25+/-5 micromoles UDP-galactose/100 g protein when grown in 0.1% glucose. The control cells had 236+/-25 micromoles UDP- glucose/100 g protein and 82+/-10 micromoles UDP-galactose/100 g protein when grown in identical medium. When we transfected the GALT-deficient cells with either the hUGP2 or GALT gene, their UDP-glucose content increased to 305+/-28 micromoles/100 g protein (hUGP2-transfected) and 210+/-13 micromoles/100 g protein (GALT-transfected), respectively. Similarly, UDP-galactose content increased to 75+/-12 micromoles/100 g protein (hUGP2-transfected) and 55+/-9 micromoles/100 g protein (GALT-transfected), respectively. Though the GALT-transfected cells grew in 0.1% galactose with little accumulation of Gal-1-P (0.2+/-0.02 mM), the hUGP2-transfected cells grew but accumulated some Gal-1-P (3.1+/-0.4 mM). We found that 2.5 mM Gal-1-P increased the apparent KM of purified hUGP2 for glucose-1-phosphate from 19.7 microM to 169 microM, without changes in apparent Vmax. The Ki of the reaction was 0.47 mM. Gal-1-P also inhibited UDP-N-acetylglucosamine pyrophosphorylase, which catalyzes the formation of UDP-N-acetylglucosamine. We conclude that intracellular concentrations of Gal-1-P found in classic galactosemia inhibit UDP-hexose pyrophosphorylases and reduce the intracellular concentrations of UDP-hexoses. Reduced Sambucus nigra agglutinin binding to glycoproteins isolated from cells with increased Gal-1-P is consistent with the resultant inhibition of glycoprotein glycosylation.
Asunto(s)
Fibroblastos/metabolismo , Galactosafosfatos/metabolismo , UTP-Hexosa-1-Fosfato Uridililtransferasa/deficiencia , Uridina Difosfato Glucosa/deficiencia , Línea Celular Transformada , Transformación Celular Viral , Células Cultivadas , Fibroblastos/efectos de los fármacos , Fibroblastos/virología , Galactosa/farmacología , Galactosafosfatos/farmacología , Glicosilación , Humanos , Cinética , Fenotipo , Virus 40 de los Simios/genética , Transfección , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , UTP-Hexosa-1-Fosfato Uridililtransferasa/genética , UTP-Hexosa-1-Fosfato Uridililtransferasa/metabolismoRESUMEN
A Chinese hamster cell line with a mutation in the UDP-glucose pyrophosphorylase (UDPG:PP) gene leading to UDP-glucose deficiency as well as a revertant cell were previously isolated. We now show that the mutant cell is 10(5) times more sensitive to the cytotoxic effect of Clostridium perfringens phospholipase C (PLC) than the revertant cell. To clarify whether there is a connection between the UDP-glucose deficiency and the hypersensitivity to C. perfringens PLC, stable transfectant cells were prepared using a wild type UDPG:PP cDNA. Clones of the mutant transfected with a construct having the insert in the sense orientation had increased their UDP-glucose level, whereas those of the revertant transfected with a UDPG:PP antisense had reduced their level of UDP-glucose compared with control clones transfected with the vector. Exposure of these two types of transfectant clones to C. perfringens PLC demonstrated that a cellular UDP-glucose deficiency causes hypersensitivity to the cytotoxic effect of this phospholipase. Further experiments with genetically engineered C. perfringens PLC variants showed that the sphingomyelinase activity and the C-domain are required for its cytotoxic effect in UDP-glucose-deficient cells.
Asunto(s)
Toxinas Bacterianas/toxicidad , Proteínas de Unión al Calcio , Clostridium perfringens/enzimología , Fosfolipasas de Tipo C/toxicidad , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , Uridina Difosfato Glucosa/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/química , Bovinos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Colina/metabolismo , Clostridium/enzimología , Cricetinae , Cricetulus , Cinética , Pulmón , Lípidos de la Membrana/biosíntesis , Fragmentos de Péptidos/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Transfección , Fosfolipasas de Tipo C/biosíntesis , Fosfolipasas de Tipo C/química , UTP-Glucosa-1-Fosfato Uridililtransferasa/deficiencia , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Uridina Difosfato Glucosa/deficienciaRESUMEN
We previously isolated a mutant cell that is the only mammalian cell reported to have a persistently low level of UDP-glucose. In this work we obtained a spontaneous revertant whose UDP-glucose level lies between those found in the wild type and the mutant cell. The activity of UDP-glucose pyrophosphorylase (UDPG:PP), the enzyme that catalyzes the formation of UDP-glucose, was in the mutant 4% and in the revertant 56% of the activity found in the wild type cell. Sequence analysis of UDPG: PP cDNAs from the mutant cell showed one missense mutation, which changes amino acid residue 115 from glycine to aspartic acid. The substituted glycine is located within the largest stretch of strictly conserved residues among eukaryotic UDPG:PPs. The analysis of the cDNAs from the revertant cell indicated the presence of an equimolar mixture of the wild type and the mutated mRNAs, suggesting that the mutation has reverted in only one of the alleles. In summary, we demonstrate that the G115D substitution in the Chinese hamster UDPG:PP dramatically impairs its enzymatic activity, thereby causing cellular UDP-glucose deficiency.
Asunto(s)
Mutación Puntual , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , Uridina Difosfato Glucosa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Cricetulus , ADN Complementario , Desoxiglucosa/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , UTP-Glucosa-1-Fosfato Uridililtransferasa/química , Uridina Difosfato Glucosa/deficienciaRESUMEN
We have previously isolated a fibroblast mutant cell with high resistance to the two Rho-modifying glucosyltransferase toxins A and B of Clostridium difficile. We demonstrate here a low level of UDP-glucose in the mutant, which explains its toxin resistance since: (i) to obtain a detectable toxin B-mediated Rho modification in lysates of mutant cells, addition of UDP-glucose was required, and it promoted the Rho modification dose-dependently; (ii) high pressure liquid chromatography analysis of nucleotide extracts of cells indicated that the level of UDP-glucose in the mutant (0.8 nmol/10(6) cells) was lower than in the wild type (3.7 nmol/10(6) cells); and (iii) sensitivity to toxin B was restored upon microinjection of UDP-glucose. Using the mutant as indicator cell we also found that the related Clostridium sordellii lethal toxin is a glucosyltransferase which requires UDP-glucose as a cofactor. Like toxin B it glucosylated 21-23-kDa proteins in cell lysates, but Rho was not a substrate for lethal toxin.