RESUMEN
Lipase maturation factor 1 (Lmf1) is an endoplasmic reticulum (ER) membrane protein involved in the posttranslational folding and/or assembly of lipoprotein lipase (LPL) and hepatic lipase (HL) into active enzymes. Mutations in Lmf1 are associated with diminished LPL and HL activities ("combined lipase deficiency") and result in severe hypertriglyceridemia in mice as well as in human subjects. Here, we investigate whether endothelial lipase (EL) also requires Lmf1 to attain enzymatic activity. We demonstrate that cells harboring a (cld) loss-of-function mutation in the Lmf1 gene are unable to generate active EL, but they regain this capacity after reconstitution with the Lmf1 wild type. Furthermore, we show that cellular EL copurifies with Lmf1, indicating their physical interaction in the ER. Finally, we determined that post-heparin phospholipase activity in a patient with the LMF1(W464X) mutation is reduced by more than 95% compared with that in controls. Thus, our study indicates that EL is critically dependent on Lmf1 for its maturation in the ER and demonstrates that Lmf1 is a required factor for all three vascular lipases, LPL, HL, and EL.
Asunto(s)
Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Hipertrigliceridemia/metabolismo , Lipasa/metabolismo , Lipoproteína Lipasa/metabolismo , Proteínas de la Membrana , Animales , Cromatografía de Afinidad , Electroporación , Retículo Endoplásmico/genética , Fibroblastos/citología , Células HEK293 , Humanos , Hipertrigliceridemia/genética , Hipertrigliceridemia/fisiopatología , Lipasa/genética , Lipoproteína Lipasa/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Plásmidos , TransfecciónRESUMEN
Lipase maturation factor 1 (LMF1) is predicted to be a polytopic protein localized to the endoplasmic reticulum (ER) membrane. It functions in the post-translational attainment of enzyme activity for both lipoprotein lipase and hepatic lipase. By using transmembrane prediction methods in mouse and human orthologs, models of LMF1 topology were constructed and tested experimentally. Employing a tagging strategy that used insertion of ectopic glycan attachment sites and terminal fusions of green fluorescent protein, we established a five-transmembrane model, thus dividing LMF1 into six domains. Three domains were found to face the cytoplasm (the amino-terminal domain and loops B and D), and the other half was oriented to the ER lumen (loops A and C and the carboxyl-terminal domain). This representative model shows the arrangement of an evolutionarily conserved domain within LMF1 (DUF1222) that is essential to lipase maturation. DUF1222 comprises four of the six domains, with the two largest ones facing the ER lumen. We showed for the first time, using several naturally occurring variants featuring DUF1222 truncations, that Lmf1 interacts physically with lipoprotein lipase and hepatic lipase and localizes the lipase interaction site to loop C within DUF1222. We discuss the implication of our results with regard to lipase maturation and DUF1222 domain structure.
Asunto(s)
Retículo Endoplásmico/metabolismo , Lipasa/metabolismo , Lipoproteína Lipasa/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Western Blotting , Línea Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Lipasa/genética , Lipoproteína Lipasa/genética , Proteínas de la Membrana/genética , Ratones , Microscopía Confocal , Modelos Biológicos , Mutación , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
OBJECTIVE: To identify metabolic derangements contributing to diabetes susceptibility in the leptin receptor-deficient obese C57BLKS/J-db/db (BKS-db) mouse strain. RESEARCH DESIGN AND METHODS: Young BKS-db mice were used to identify metabolic pathways contributing to the development of diabetes. Using the diabetes-resistant B6-db strain as a comparison, in vivo and in vitro approaches were applied to identify metabolic and molecular differences between the two strains. RESULTS: Despite higher plasma insulin levels, BKS-db mice exhibit lower lipogenic gene expression, rate of lipogenesis, hepatic triglyceride and glycogen content, and impaired insulin suppression of gluconeogenic genes. Hepatic insulin receptor substrate (IRS)-1 and IRS-2 expression and insulin-stimulated Akt-phosphorylation are decreased in BKS-db primary hepatocytes. Hyperinsulinemic-euglycemic clamp studies indicate that in contrast to hepatic insulin resistance, skeletal muscle is more insulin sensitive in BKS-db than in B6-db mice. We also demonstrate that elevated plasma triglyceride levels in BKS-db mice are associated with reduced triglyceride clearance due to lower lipase activities. CONCLUSIONS: Our study demonstrates the presence of metabolic derangements in BKS-db before the onset of beta-cell failure and identifies early hepatic insulin resistance as a component of the BKS-db phenotype. We propose that defects in hepatic insulin signaling contribute to the development of diabetes in the BKS-db mouse strain.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Hepatocitos/metabolismo , Resistencia a la Insulina/genética , Insulina/metabolismo , Hígado/metabolismo , Análisis de Varianza , Animales , Diabetes Mellitus Tipo 2/genética , Ácidos Grasos/metabolismo , Expresión Génica , Gluconeogénesis/genética , Hepatocitos/citología , Insulina/genética , Lipasa/metabolismo , Lipogénesis/genética , Ratones , Ratones Endogámicos C57BL , Obesidad/genética , Obesidad/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
Tandem affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in Chinese hamster ovary cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, five function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase were also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57, and glucosidase II, followed by repeated posttranslational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding and may help direct its eventual degradation.
Asunto(s)
Lipasa/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cromatografía de Afinidad , Cricetinae , Cricetulus , Ditiotreitol/farmacología , Chaperón BiP del Retículo Endoplásmico , Humanos , Lipasa/genética , Lipasa/aislamiento & purificación , Modelos Biológicos , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Modificación Traduccional de las Proteínas , Procesamiento Proteico-Postraduccional , Proteoma/genética , Proteoma/aislamiento & purificación , Proteoma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en Tándem , TransfecciónRESUMEN
Hypertriglyceridemia is a hallmark of many disorders, including metabolic syndrome, diabetes, atherosclerosis and obesity. A well-known cause is the deficiency of lipoprotein lipase (LPL), a key enzyme in plasma triglyceride hydrolysis. Mice carrying the combined lipase deficiency (cld) mutation show severe hypertriglyceridemia owing to a decrease in the activity of LPL and a related enzyme, hepatic lipase (HL), caused by impaired maturation of nascent LPL and hepatic lipase polypeptides in the endoplasmic reticulum (ER). Here we identify the gene containing the cld mutation as Tmem112 and rename it Lmf1 (Lipase maturation factor 1). Lmf1 encodes a transmembrane protein with an evolutionarily conserved domain of unknown function that localizes to the ER. A human subject homozygous for a deleterious mutation in LMF1 also shows combined lipase deficiency with concomitant hypertriglyceridemia and associated disorders. Thus, through its profound effect on lipase activity, LMF1 emerges as an important candidate gene in hypertriglyceridemia.
Asunto(s)
Codón sin Sentido , Predisposición Genética a la Enfermedad , Hipertrigliceridemia/genética , Lipoproteína Lipasa/genética , Animales , Retículo Endoplásmico , Humanos , Lipoproteína Lipasa/química , Ratones , Estructura Terciaria de ProteínaRESUMEN
Among three lipases in the lipase gene family, hepatic lipase (HL), lipoprotein lipase, and pancreatic lipase, HL exhibits the lowest intracellular specific activity (i.e. minimal amounts of catalytic activity accompanied by massive amounts of inactive lipase mass in the endoplasmic reticulum (ER)). In addition, HL has a distinctive sedimentation profile, where the inactive mass overlaps the region containing active dimeric HL and trails into progressively larger molecular forms. Eventually, at least half of the HL inactive mass in the ER reaches an active, dimeric conformation (t(1/2) = 2 h) and is rapidly secreted. The remaining inactive mass is degraded. HL maturation occurs in the ER and is strongly dependent on binding to calnexin in the early co-/post-translational stages. Later stages of HL maturation occur without calnexin assistance, although inactive HL at all stages appears to be associated in distinct complexes with other ER proteins. Thus, unlike other lipases in the gene family, HL maturation is the rate-limiting step in its secretion as a functional enzyme.
Asunto(s)
Retículo Endoplásmico/enzimología , Lipasa/química , Hígado/enzimología , Animales , Western Blotting , Células CHO , Calnexina/química , Línea Celular , Centrifugación por Gradiente de Densidad , Cricetinae , Reactivos de Enlaces Cruzados/farmacología , Cicloheximida/farmacología , ADN Complementario/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Indolizinas/farmacología , Lipasa/metabolismo , Pruebas de Precipitina , Conformación Proteica , Pliegue de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Sacarosa/farmacología , Factores de Tiempo , TransfecciónRESUMEN
The maturation of lipoprotein lipase (LPL) into a catalytically active enzyme was believed to occur only after its transport from the endoplasmic reticulum (ER) to the Golgi apparatus. To test this hypothesis, LPL located in these two subcellular compartments was separated and compared. Heparin affinity chromatography resolved low affinity, inactive LPL displaying ER characteristics from a high affinity, active fraction exhibiting both ER and Golgi forms. The latter forms were further separated by beta-ricin chromatography and were found to have comparable activities per unit of LPL mass. Thus, LPL must reach a functional conformation in the ER. Active LPL, regardless of its cellular location, exhibited the expected dimer conformation. However, inactive LPL, found only in the ER, was highly aggregated. Kinetic analysis indicated a concurrent formation of LPL dimer and aggregate and indicated that the two forms have dissimilar fates. Whereas the dimer remained stable even when confined to the ER, the aggregate was degraded. Degradation rates were not affected by proteasomal or lysosomal inhibitors but were markedly reduced by ATP depletion. Lowering the redox potential in the ER by dithiothreitol caused the dimer to associate with calnexin, BiP, and protein-disulfide isomerase to form large, inactive complexes; dithiothreitol removal induced complex dissociation with restoration of the functional LPL dimer. In contrast, the LPL aggregate was only poorly associated with ER chaperones, appearing to be trapped in an irreversible, inactive conformation destined for ER degradation.
Asunto(s)
Retículo Endoplásmico/enzimología , Aparato de Golgi/metabolismo , Proteínas de Choque Térmico , Heparina/metabolismo , Lipoproteína Lipasa/química , Lipoproteína Lipasa/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Western Blotting , Células CHO , Proteínas de Unión al Calcio/metabolismo , Calnexina , Proteínas Portadoras/metabolismo , Línea Celular , Células Cultivadas , Centrifugación por Gradiente de Densidad , Cricetinae , ADN Complementario/metabolismo , Dimerización , Disulfuros , Chaperón BiP del Retículo Endoplásmico , Glicósido Hidrolasas/metabolismo , Humanos , Lisosomas/metabolismo , Chaperonas Moleculares/metabolismo , Oxidación-Reducción , Pruebas de Precipitina , Unión Proteica , Conformación Proteica , Proteína Disulfuro Isomerasas/metabolismo , Pliegue de Proteína , Solubilidad , Factores de Tiempo , TransfecciónRESUMEN
Lipoprotein lipase (LpL) hydrolyzes triglycerides of circulating lipoproteins while bound as homodimers to endothelial cell surface heparan sulfate proteoglycans. This primarily occurs in the capillary beds of muscle and adipose tissue. By creating a mouse line that expresses covalent dimers of heparin-binding deficient LpL (hLpLHBM-Dimer) in muscle, we confirmed in vivo that linking two LpL monomers in a head to tail configuration creates a functional LpL. The hLpLHBM-Dimer transgene produced abundant activity and protein in muscle, and the LpL was the expected size of a dimer (approximately 110 kDa). Unlike the heparin-binding mutant monomer, hLpLHBM-Dimer had the same stability as nonmutated LpL. The hLpLHBM-Dimer transgene prevented the neonatal demise of LpL knockout mice; however, these mice were hypertriglyceridemic. Postheparin plasma LpL activity was lower than expected with the robust expression in muscle and was no longer covalently linked. Studies in transfected cells showed that Chinese hamster lung cells, but not COS cells, also degraded tandem repeated LpL into monomers. Thus, although muscle can synthesize tethered, dimeric LpL, efficient production of this enzyme leading to secretion, and physiological function appears to favor secretion of a noncovalent dimer composed of monomeric subunits.