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Exosomes are small vesicles secreted from multivesicular bodies, which are able to stimulate the immune system leading to tumour cell eradication. We have analysed lipids of exosomes secreted either upon stimulation from rat mast cells (RBL-2H3 cells), or constitutively from human dendritic cells. As compared with parent cells, exosomes displayed an enrichment in sphingomyelin, but not in cholesterol. Phosphatidylcholine content was decreased, but an enrichment was noted in disaturated molecular species as in phosphatidylethanolamines. Lyso(bis)phosphatidic acid was not enriched in exosomes as compared with cells. Fluorescence anisotropy demonstrated an increase in exosome-membrane rigidity from pH 5 to 7, suggesting their membrane reorganization between the acidic multivesicular body compartment and the neutral outer cell medium. NMR analysis established a bilayer organization of exosome membrane, and ESR studies using 16-doxyl stearic acid demonstrated a higher flip-flop of lipids between the two leaflets as compared with plasma membrane. In addition, the exosome membrane exhibited no asymmetrical distribution of phosphatidylethanolamines. Therefore exosome membrane displays a similar content of the major phospholipids and cholesterol, and is organized as a lipid bilayer with a random distribution of phosphatidylethanolamines. In addition, we observed tight lipid packing at neutral pH and a rapid flip-flop between the two leaflets of exosome membranes. These parameters could be used as a hallmark of exosomes.

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The effect of high-density lipoprotein (HDL) in protecting against atherosclerosis is usually attributed to its role in 'reverse cholesterol transport'. In this process, HDL particles mediate the efflux and the transport of cholesterol from peripheral cells to the liver for further metabolism and bile excretion. Thus, cell-surface receptors for HDL on hepatocytes are chief partners in the regulation of cholesterol homeostasis. A high-affinity HDL receptor for apolipoprotein A-I (apoA-I) was previously identified on the surface of hepatocytes. Here we show that this receptor is identical to the beta-chain of ATP synthase, a principal protein complex of the mitochondrial inner membrane. Different experimental approaches confirm this ectopic localization of components of the ATP synthase complex and the presence of ATP hydrolase activity at the hepatocyte cell surface. Receptor stimulation by apoA-I triggers the endocytosis of holo-HDL particles (protein plus lipid) by a mechanism that depends strictly on the generation of ADP. We confirm this effect on endocytosis in perfused rat liver ex vivo by using a specific inhibitor of ATP synthase. Thus, membrane-bound ATP synthase has a previously unsuspected role in modulating the concentrations of extracellular ADP and is regulated by a principal plasma apolipoprotein.

The impact of shift work on cardiovascular disease (CVD) risk factors and metabolic syndrome are not yet completely understood. The objectives of this study were to evaluate the impact of shift work on metabolic syndrome according to two different definitions in a population of strictly rotating shift workers (3x8 h) compared to paired counterparts working only day hours, and to study whether shift work itself is a determinant of metabolic syndrome after taking into account a large panel of confusing factors. We conducted a cross-sectional study comparing 98 strictly rotating shift workers to 100 regular day-workers (all subjects had a long experience of their working rhythms) within the same petrochemical plant. Clinical, behavioral, occupational, and biological data were collected, and a detailed nutritional investigation was done. Shift and day workers were comparable in terms of major CVD factors, and both had a 10 yr Framingham risk scoring of 11%. Shift workers reported an increased job strain and higher total and at-work physical activity. Alterations in metabolic parameters were evident with a rise in triglycerides, free fatty acids, and gamma glutamyl transpeptidase and lower HDL-cholesterol. Multiple logistic regression analysis demonstrated that shift work was associated with occurrence of metabolic syndrome, as defined by the National Cholesterol Education Program-ATPIII criteria, OR: 2.38 (1.13-4.98), but not using the more recent score from the International Diabetes Federation, which gives a major emphasis on abdominal obesity. Total energy intake and contributions of the major nutrients did not differ between the two groups, with the notable exception of saturated lipids (+10% in shift workers). Meal distribution was clearly different: energy intake was more fractionated within the day, with a lesser contribution of breakfast and lunch but with increased intakes during intermediate light meals, particularly in the afternoon and night. Multivariate analyses were performed to test for the influence of dietary rhythms on the development of an NCEP-ATPIII metabolic syndrome. Dietary intakes at breakfast and during intermediate light meals appear to be "protective" against metabolic syndrome, while a high load at dinner favors its occurrence. A high intake at lunch is particularly deleterious to shift workers. However, in all tested models, shift work remained significantly associated with metabolic syndrome, after taking into account potential covariates like job strain, physical activity, quantitative dietary parameters, and meal distribution. A specific follow-up of shift workers should be recommended to occupational physicians.

Vgamma9Vdelta2 T lymphocytes, a major gammadelta T lymphocyte subset in humans, display cytolytic activity against various tumor cells upon recognition of yet uncharacterized structures. Here, we show that an entity related to the mitochondrial F1-ATPase is expressed on tumor cell surface and promotes tumor recognition by Vgamma9Vdelta2 T cells. When immobilized, purified F1-ATPase induces selective activation of this lymphocyte subset. The Vgamma9Vdelta2 T cell receptors (TCR) and the F1-ATPase also bind a delipidated form of apolipoprotein A-I (apo A-I), as demonstrated by surface plasmon resonance. Moreover, the presence of apo A-I in the culture medium is required for optimal activation of Vgamma9Vdelta2 T cells by tumors expressing F1-ATPase. This study thus describes an unanticipated tumor recognition mechanism by Vgamma9Vdelta2 lymphocytes and a possible link between gammadelta T cell immunity and lipid metabolism.

SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr 317 were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation.The GTPase Ras is a crucial signaling relay of receptor tyrosine kinases, and the mechanisms controlling its activation by growth factors appear well understood. For example, the activated epidermal growth factor (EGF) 1 receptor (EGFR) autophosphorylates on tyrosine residues, which creates docking sites for phosphotyrosine-binding domains (e.g. SH2) of adaptor proteins, including Grb2 and Shc. Because Grb2 is constitutively associated with Sos, a guanine nucleotide exchange factor of Ras, the binding of Grb2 to phosphorylated EGFR results in the recruitment of Sos to the plasma membrane and has been proposed as a model for activation of membrane-bound Ras. Once activated, Ras can stimulate several effectors, notably the protein Ser/Thr kinase Raf1 and the downstream activated mitogen-activated protein kinases Erk1/2, major regulators of cell proliferation, differentiation, and survival (1). Although this pathway has reached a canonical status, the docking protein Gab1 and one of its binding partners, SHP2, were recently found to participate in Ras/Erk activation in response to EGF and other growth factors (2-5). SHP2 is a protein-tyrosine phosphatase with two SH2 domains and phosphorylation sites with affinity for Grb2 SH2 domains. Its catalytic activity is stimulated by engagement of its SH2 domains with specific phosp...