RESUMEN
The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate-screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N-methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing "jackpot" clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in-culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Pichia/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Acuaporina 4/biosíntesis , Acuaporina 4/genética , Biotecnología , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Fosfatidiletanolamina N-Metiltransferasa/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa/genética , Pichia/genética , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Bile salts are potent detergents and can disrupt cellular membranes, which causes cholestasis and hepatocellular injury. However, the mechanism for the resistance of the canalicular membrane against bile salts is not clear. Phosphatidylethanolamine (PE) is converted to phosphatidylcholine (PC) in the liver by phosphatidylethanolamine N-methyltransferase (PEMT). In this study, to investigate the effect of PEMT expression on the resistance to bile salts, we established an LLC-PK1 cell line stably expressing PEMT. By using enzymatic assays, we showed that the expression of PEMT increased the cellular PC content, lowered the PE content, but had no effect on the sphingomyelin content. Consequently, PEMT expression led to reductions in PE/PC and sphingomyelin/PC ratios. Mass spectrometry demonstrated that PEMT expression increased the levels of PC species containing longer acyl chains and almost all ether-linked PC species. PEMT expression enhanced the resistance to duramycin and lysenin, suggesting decreased ratios of PE and sphingomyelin in the apical membrane, respectively. In addition, SEM revealed that PEMT expression increased the diameter of microvilli. The expression of PEMT resulted in reduced resistance to unconjugated bile salts, but surprisingly in increased resistance to conjugated bile salts, which might be attributable to modifications of the phospholipid composition and/or structure in the apical membrane. Because most bile salts exist as conjugated forms in the bile canaliculi, PEMT may be important in the protection of hepatocytes from bile salts and in cholestatic liver injury.
Asunto(s)
Ácidos y Sales Biliares/farmacología , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Fosfolípidos/metabolismo , Esfingomielinas/metabolismo , Animales , Bacteriocinas/farmacología , Canalículos Biliares/metabolismo , Membrana Celular/efectos de los fármacos , Citotoxinas/farmacología , Resistencia a Medicamentos , Células LLC-PK1 , Microvellosidades/efectos de los fármacos , Microvellosidades/metabolismo , Péptidos/farmacología , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa/biosíntesis , Fosfatidiletanolaminas/metabolismo , PorcinosRESUMEN
The endoplasmic reticulum (ER) is the main site of protein and lipid synthesis, membrane biogenesis, xenobiotic detoxification and cellular calcium storage, and perturbation of ER homeostasis leads to stress and the activation of the unfolded protein response. Chronic activation of ER stress has been shown to have an important role in the development of insulin resistance and diabetes in obesity. However, the mechanisms that lead to chronic ER stress in a metabolic context in general, and in obesity in particular, are not understood. Here we comparatively examined the proteomic and lipidomic landscape of hepatic ER purified from lean and obese mice to explore the mechanisms of chronic ER stress in obesity. We found suppression of protein but stimulation of lipid synthesis in the obese ER without significant alterations in chaperone content. Alterations in ER fatty acid and lipid composition result in the inhibition of sarco/endoplasmic reticulum calcium ATPase (SERCA) activity and ER stress. Correcting the obesity-induced alteration of ER phospholipid composition or hepatic Serca overexpression in vivo both reduced chronic ER stress and improved glucose homeostasis. Hence, we established that abnormal lipid and calcium metabolism are important contributors to hepatic ER stress in obesity.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Metabolismo de los Lípidos , Hígado/patología , Obesidad/metabolismo , Estrés Fisiológico , Animales , Retículo Endoplásmico/patología , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Leptina/deficiencia , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/enzimología , Obesidad/patología , Obesidad/fisiopatología , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa/genética , Fosfatidiletanolaminas/metabolismo , Biosíntesis de Proteínas , Proteómica , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Delgadez/metabolismoRESUMEN
In eukaryotes, S-adenosyl-L-homocysteine hydrolase (Sah1) offers a single way for degradation of S-adenosyl-L-homocysteine, a product and potent competitive inhibitor of S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases. De novo phosphatidylcholine (PC) synthesis requires three AdoMet-dependent methylation steps. Here we show that down-regulation of SAH1 expression in yeast leads to accumulation of S-adenosyl-L-homocysteine and decreased de novo PC synthesis in vivo. This decrease is accompanied by an increase in triacylglycerol (TG) levels, demonstrating that Sah1-regulated methylation has a major impact on cellular lipid homeostasis. TG accumulation is also observed in cho2 and opi3 mutants defective in methylation of phosphatidylethanolamine to PC, confirming that PC de novo synthesis and TG synthesis are metabolically coupled through the efficiency of the phospholipid methylation reaction. Indeed, because both types of lipids share phosphatidic acid as a precursor, we find in cells with down-regulated Sah1 activity major alterations in the expression of the INO1 gene as well as in the localization of Opi1, a negative regulatory factor of phospholipid synthesis, which binds and is retained in the endoplasmic reticulum membrane by phosphatidic acid in conjunction with VAMP/synaptobrevin-associated protein, Scs2. The addition of homocysteine, by the reversal of the Sah1-catalyzed reaction, also leads to TG accumulation in yeast, providing an attractive model for the role of homocysteine as a risk factor of atherosclerosis in humans.
Asunto(s)
Adenosilhomocisteinasa/biosíntesis , Aterosclerosis/enzimología , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Fosfatidilcolinas/biosíntesis , Saccharomyces cerevisiae/enzimología , Triglicéridos/biosíntesis , Adenosilhomocisteinasa/genética , Aterosclerosis/genética , Regulación hacia Abajo/genética , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/genética , Regulación Enzimológica de la Expresión Génica/genética , Regulación Fúngica de la Expresión Génica/genética , Homeostasis/genética , Homocisteína/genética , Homocisteína/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Metilación , Mio-Inositol-1-Fosfato Sintasa/biosíntesis , Mio-Inositol-1-Fosfato Sintasa/genética , Fosfatidilcolinas/genética , Fosfatidiletanolamina N-Metiltransferasa/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Factores de Riesgo , S-Adenosilhomocisteína/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Triglicéridos/genéticaRESUMEN
BACKGROUND: Some humans fed a low-choline diet develop hepatosteatosis, liver and muscle damage, and lymphocyte apoptosis. The risk of developing such organ dysfunction is increased by the presence of single-nucleotide polymorphisms (SNPs) in genes involved in folate and choline metabolism. OBJECTIVE: We investigated whether these changes that occur in the expression of many genes when humans are fed a low-choline diet differ between subjects who develop organ dysfunction and those who do not. We also investigated whether expression changes were dependent on the presence of the SNPs of interest. DESIGN: Thirty-three subjects aged 20-67 y were fed for 10 d a baseline diet containing the recommended adequate intake of choline. They then were fed a low-choline diet for up to 42 d or until they developed organ dysfunction. Blood was collected at the end of each phase, and peripheral lymphocytes were isolated and used for genotyping and for gene expression profiling with the use of microarray hybridization. RESULTS: Feeding a low-choline diet changed the expression of 259 genes, and the profiles of subjects who developed and those who did not develop signs of organ dysfunction differed. Group clustering and gene ontology analyses found that the diet-induced changes in gene expression profiles were significantly influenced by the SNPs of interest and that the gene expression phenotype of the variant gene carriers differed significantly even with the baseline diet. CONCLUSION: These findings support our hypothesis that a person's susceptibility to organ dysfunction when fed a low-choline diet is modulated by specific SNPs in genes involved in folate and choline metabolism.
Asunto(s)
Deficiencia de Colina/sangre , Deficiencia de Colina/genética , Linfocitos/fisiología , Adulto , Anciano , Colina/administración & dosificación , Deficiencia de Colina/enzimología , Colina-Deshidrogenasa/biosíntesis , Colina-Deshidrogenasa/genética , Análisis por Conglomerados , ADN/química , ADN/genética , Femenino , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Linfocitos/enzimología , Linfocitos/metabolismo , Masculino , Metilenotetrahidrofolato Deshidrogenasa (NADP)/biosíntesis , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidiletanolamina N-Metiltransferasa/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa/genética , Polimorfismo de Nucleótido SimpleRESUMEN
OBJECTIVE: To explore the mechanism of cell proliferation inhibition by transfecting phosphatidylethanolamine N-methyltransferase 2 gene (PEMT2). METHODS: The expression and translocation of different isoforms of protein kinase C (PKC) in cells were observed with immunocytochemistry and Western blot techniques. The content of diacylglycerol (DAG) was analyzed with high performance thin layer chromatography (HPTLC) technique. RESULTS: Transfection of PEMT2 can inhibit the expression of cPKC alpha, but obviously promotes the expression and translocation from cytosol to plasma membrane of cPKC beta2. At the same time, the content of DAG was decreased in the transfected cells. Expression and translocation of other PKC isoforms were not changed by PEMT2 transfection. CONCLUSION: Effects of overexpression of PEMT2 on the expression and translocation of different PKC isoforms might be related to the mechanism of cell proliferation inhibition and apoptosis induced by transfecting PEMT2.