RESUMEN
Gaucher's disease (GD), an inherited metabolic disorder caused by mutations in the glucocerebrosidase gene (GBA), is the most common lysosomal storage disease. Heterozygous mutations in GBA are a major risk factor for Parkinson's disease. GD is divided into three clinical subtypes based on the absence (type 1) or presence (types 2 and 3) of neurological signs. Type 1 GD was the first lysosomal storage disease (LSD) for which enzyme therapy became available, and although infusions of recombinant glucocerebrosidase (GCase) ameliorate the systemic effects of GD, the lack of efficacy for the neurological manifestations, along with the considerable expense and inconvenience of enzyme therapy for patients, renders the search for alternative or complementary therapies paramount. Glucosylceramide and glucosylsphingosine accumulation in the brain leads to massive neuronal loss in patients with neuronopathic GD (nGD) and in nGD mouse models. However, the mode of neuronal death is not known. Here, we show that modulating the receptor-interacting protein kinase-3 (Ripk3) pathway markedly improves neurological and systemic disease in a mouse model of GD. Notably, Ripk3 deficiency substantially improved the clinical course of GD mice, with increased survival and motor coordination and salutary effects on cerebral as well as hepatic injury.
Asunto(s)
Encéfalo/patología , Muerte Celular/fisiología , Enfermedad de Gaucher/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Western Blotting , Fluoresceínas , Enfermedad de Gaucher/metabolismo , Enfermedad de Gaucher/patología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Destreza Motora/fisiología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficienciaRESUMEN
Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme. prGCD also displays a level of biological activity similar to that of Cerezyme produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease.
Asunto(s)
Enfermedad de Gaucher/tratamiento farmacológico , Glucosilceramidasa/metabolismo , Glucosilceramidasa/uso terapéutico , Polisacáridos/metabolismo , Animales , Western Blotting , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Cristalografía por Rayos X , Daucus carota/citología , Daucus carota/enzimología , Daucus carota/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Enfermedad de Gaucher/enzimología , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Humanos , Macrófagos/metabolismo , Masculino , Manosa/química , Manosa/metabolismo , Ratones , Ratones Endogámicos ICR , Polisacáridos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéuticoRESUMEN
A new series of hybrid PDMP analogues, based both on PDMP and styryl analogues of natural ceramide, has been synthesized from D-serine. The synthetic route was developed such that future introduction of different aryl groups is straightforward. Biological evaluation, both in vitro on rat liver Golgi fractions as well as in HEK-293 and COS-7 cells, revealed two lead compounds with comparable inhibitory potency as PDMP, which could be elaborated to more potent inhibitors.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glucosiltransferasas/antagonistas & inhibidores , Aparato de Golgi/efectos de los fármacos , Morfolinas/farmacología , Animales , Células COS , Línea Celular , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Aparato de Golgi/enzimología , Humanos , Hígado/efectos de los fármacos , Hígado/enzimología , Estructura Molecular , Morfolinas/química , Ratas , Relación Estructura-Actividad , Fracciones Subcelulares/enzimologíaRESUMEN
Overexpression of upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene (LAG1), selectively induces the synthesis of stearoyl-containing sphingolipids in mammalian cells (Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Sullards, M. C., Merrill, A. H. Jr., and Futerman, A. H. (2002) J. Biol. Chem. 277, 35642-35649). Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag1p motif, previously characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH). We now report that two additional members of this family regulate the synthesis of (dihydro)ceramides with specific fatty acid(s) when overexpressed in human embryonic kidney 293T cells. TRH1 or TRH4-overexpression elevated [3H](dihydro)ceramide synthesis from l-[3-3H]serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B1 (FB1). Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4 overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH1 overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB1. A similar fatty acid specificity was obtained upon analysis of (dihydro)ceramide synthase activity in vitro using various fatty acyl-CoA substrates, although in a FB1-sensitive manner. Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH1-overexpressing cells. These findings lend support to our hypothesis (Venkataraman, K., and Futerman, A. H. (2002) FEBS Lett. 528, 3-4) that Lag1p family members regulate (dihydro)ceramide synthases responsible for production of sphingolipids containing different fatty acids.
Asunto(s)
Acilcoenzima A/metabolismo , Ceramidas/biosíntesis , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Animales , Células COS , Línea Celular , Cromatografía Liquida , ADN Complementario/metabolismo , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/metabolismo , Fumonisinas/farmacología , Regulación de la Expresión Génica , Humanos , Cinética , Proteínas de la Membrana/química , Ratones , Microscopía Fluorescente , Familia de Multigenes , Ácidos Palmíticos/metabolismo , Estructura Terciaria de Proteína , Esfingolípidos/metabolismo , Esfingosina N-Aciltransferasa , Factores de Tiempo , Distribución Tisular , Transfección , Proteínas Supresoras de TumorRESUMEN
Gangliosides are found at high levels in neuronal tissues where they play a variety of important functions. In the gangliosidoses, gangliosides accumulate because of defective activity of the lysosomal proteins responsible for their degradation, usually resulting in a rapidly progressive neurodegenerative disease. However, the molecular mechanism(s) leading from ganglioside accumulation to neurodegeneration is not known. We now examine the effect of ganglioside GM2 accumulation in a mouse model of Sandhoff disease (one of the GM2 gangliosidoses), the Hexb-/- mouse. Microsomes from Hexb-/- mouse brain showed a significant reduction in the rate of Ca2+-uptake via the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), which was prevented by feeding Hexb-/- mice with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glycolipid synthesis that reduces GM2 storage. Changes in SERCA activity were not due to transcriptional regulation but rather because of a decrease in Vmax. Moreover, exogenously added GM2 had a similar effect on SERCA activity. The functional significance of these findings was established by the enhanced sensitivity of neurons cultured from embryonic Hexb-/- mice to cell death induced by thapsigargin, a specific SERCA inhibitor, and by the enhanced sensitivity of Hexb-/- microsomes to calcium-induced calcium release. This study suggests a mechanistic link among GM2 accumulation, reduced SERCA activity, and neuronal cell death, which may be of significance for delineating the neuropathophysiology of Sandhoff disease.