RESUMEN
Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare recessive disease characterized by near absence of adipose tissue and severe insulin resistance. In most cases, BSCL is due to loss-of-function mutations in the genes encoding either seipin of unknown function or 1-acyl-glycerol-3-phosphate O-acyltransferase 2 (AGPAT2) which catalyses the formation of phosphatidic acid from lysophosphatidic acid. We studied the lipid profile of lymphoblastoid cell-lines from 20 BSCL patients with null mutations in the genes encoding either seipin (n=12) or AGPAT2 (n=8) in comparison to nine control cell-lines. In seipin deficient cells, we observed alterations in the pattern of lipid droplets which were decreased in size and increased in number as compared to control cells. We also observed alterations in the triglycerides content as well as in the fatty acid composition from triglycerides and phosphatidylethanolamine, with an increased proportion of saturated fatty acids at the expense of the corresponding monounsaturated fatty acids, reflecting a defect in Delta9-desaturase activity. In AGPAT2 deficient cells, no specific alterations in lipid droplet pattern nor in fatty acid composition was observed but the cellular level of lysophosphatidic acid was increased as compared to that of control and seipin deficient cells. These results indicate that seipin like AGPAT2 is involved in lipid metabolism but exerts a different function. Seipin intervenes at a proximal step in triglycerides and phospholipids biosynthesis being involved in the pathway that links fatty acid Delta9 desaturation to lipid droplet formation. These findings provide new insights into how seipin deficiency causes severe lipodystrophy.
Asunto(s)
Ácidos Grasos Insaturados/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/deficiencia , Metabolismo de los Lípidos , Lipodistrofia Generalizada Congénita/patología , Mutación , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferasa/metabolismo , Adolescente , Adulto , Línea Celular Transformada , Niño , Preescolar , Ácidos Grasos Insaturados/química , Femenino , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Humanos , Lactante , Lípidos/análisis , Lípidos/química , Lipodistrofia Generalizada Congénita/genética , Lipodistrofia Generalizada Congénita/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Linfocitos/ultraestructura , Masculino , Microscopía Confocal , Microscopía Electrónica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estearoil-CoA Desaturasa/metabolismo , Triglicéridos/metabolismo , Adulto JovenRESUMEN
Previous studies have shown that rotavirus virions, a major cause of infantile diarrhea, assemble within small intestinal enterocytes and are released at the apical pole without significant cell lysis. In contrast, for the poorly differentiated kidney epithelial MA 104 cells, which have been used extensively to study rotavirus assembly, it has been shown that rotavirus is released by cell lysis. The subsequent discovery that rotavirus particles associate with raft-type membrane microdomains (RTM) in Caco-2 cells provided a simple explanation for rotavirus polarized targeting. However, the results presented here, together with those recently published by another group, demonstrate that rotavirus also associates with RTM in MA 104 cells, thus indicating that a simple interaction of rotavirus with rafts is not sufficient to explain its apical targeting in intestinal cells. In the present study, we explore the possibility that RTM may have distinct physicochemical properties that may account for the differences observed in the rotavirus cell cycle between MA 104 and Caco-2 cells. We show here that VP4 association with rafts is sensitive to cholesterol extraction by methyl-beta-cyclodextrin treatment in MA 104 cells and insensitive in Caco-2 cells. Using the VP4 spike protein as bait, VP4-enriched raft subsets were immunopurified. They contained 10 to 15% of the lipids present in total raft membranes. We found that the nature and proportion of phospholipids and glycosphingolipids were different between the two cell lines. We propose that this raft heterogeneity may support the cell type dependency of virus assembly and release.
Asunto(s)
Proteínas de la Cápside/metabolismo , Membrana Celular/metabolismo , Rotavirus/fisiología , Animales , Células CACO-2/metabolismo , Células CACO-2/virología , Línea Celular , Membrana Celular/química , Relación Dosis-Respuesta a Droga , Glicoesfingolípidos/análisis , Glicoesfingolípidos/aislamiento & purificación , Humanos , Fosfolípidos/análisis , Fosfolípidos/aislamiento & purificación , Unión Proteica/efectos de los fármacos , Especificidad de la Especie , Ensamble de Virus , beta-Ciclodextrinas/farmacologíaAsunto(s)
Polaridad Celular , Aparato de Golgi/metabolismo , Intestinos/virología , Microdominios de Membrana/metabolismo , Rotavirus/metabolismo , Ensamble de Virus , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Intestinos/citología , Rotavirus/patogenicidad , Infecciones por Rotavirus/virologíaRESUMEN
Rotavirus assembly is a multistep process that requires the successive association of four major structural proteins in three concentric layers. It has been assumed until now that VP4, the most external viral protein that forms the spikes of mature virions, associates with double-layer particles within the endoplasmic reticulum (ER) in conjunction with VP7 and with the help of a nonstructural protein, NSP4. VP7 and NSP4 are two glycosylated proteins. However, we recently described a strong association of VP4 with raft-type membrane microdomains, a result that makes the ER a highly questionable site for the final assembly of rotavirus, since rafts are thought to be absent from this compartment. In this study, we used tunicamycin (TM), a drug known to block the first step of protein N glycosylation, as a tool to dissect rotavirus assembly. We show that, as expected, TM blocks viral protein glycosylation and also decreases virus infectivity. In the meantime, viral particles were blocked as enveloped particles in the ER. Interestingly, TM does not prevent the targeting of VP4 to the cell surface nor its association with raft membranes, whereas the infectivity associated with the raft fractions strongly decreased. VP4 does not colocalize with the ER marker protein disulfide-isomerase even when viral particles were blocked by TM in this compartment. These results strongly support a primary role for raft membranes in rotavirus final assembly and the fact that VP4 assembly with the rest of the particle is an extrareticular event.
Asunto(s)
Proteínas de la Cápside/metabolismo , Polaridad Celular , Retículo Endoplásmico/virología , Rotavirus/fisiología , Ensamble de Virus , Células CACO-2 , Humanos , Microdominios de Membrana/virología , Tunicamicina/farmacología , Virión/metabolismoRESUMEN
Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.
Asunto(s)
Células CACO-2/virología , Proteínas de la Cápside , Cápside/metabolismo , Microdominios de Membrana/metabolismo , Rotavirus/patogenicidad , Ensamble de Virus , Membrana Celular/efectos de los fármacos , Resistencia a Medicamentos , Humanos , Microdominios de Membrana/efectos de los fármacos , Octoxinol/farmacología , Rotavirus/metabolismoRESUMEN
Recent studies suggest that rafts are involved in numerous cell functions, including membrane traffic and signaling. Here we demonstrate, using a polyoxyethylene ether Brij 98, that detergent-insoluble microdomains possessing the expected biochemical characteristics of rafts are present in the cell membrane at 37 degrees C. After extraction, these microdomains are visualized as membrane vesicles with a mean diameter of approximately 70 nm. These findings provide further evidence for the existence of rafts under physiological conditions and are the basis of a new isolation method allowing more accurate analyses of raft structure. We found that main components of T cell receptor (TCR) signal initiation machinery, i.e. TCR-CD3 complex, Lck and ZAP-70 kinases, and CD4 co-receptor are constitutively partitioned into a subset of rafts. Functional studies in both intact cells and isolated rafts showed that upon ligation, TCR initiates the signaling in this specialized raft subset. Our data thus strongly indicate an important role of rafts in organizing TCR early signaling pathways within small membrane microdomains, both prior to and following receptor engagement, for efficient TCR signal initiation upon stimulation.