28 results on '"Nauze M"'
Search Results
2. Etude de l’absorption intestinale de lipides dans un modèle murin surexprimant au niveau intestinal le Scavenger Receptor Class B type I : SR-BI
- Author
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Bietrix, F, Yan, D, Nauze, M, Rolland, C, Schaak, S, Martin, Pascal G.P., Pineau, Thierry, Barbaras, R, Perret, B, Tercet, F, Collet, Xavier, Inconnu, Unité de recherche Pharmacologie-Toxicologie (UPT), Institut National de la Recherche Agronomique (INRA), Département Santé Animale (DEPT SA), and ProdInra, Migration
- Subjects
[SDV] Life Sciences [q-bio] ,[SDV]Life Sciences [q-bio] ,ComputingMilieux_MISCELLANEOUS - Abstract
National audience
- Published
- 2004
3. The nucleotide receptor P2Y13 is a key regulator of hepatic high-density lipoprotein (HDL) endocytosis.
- Author
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Jacquet, S, Malaval, C, Martinez, Laurent O, Sak, Katrin, Rolland, C, Perez, C, Nauze, M, Champagne, E, Tercé, F, Gachet, Christian, Perret, B, Collet, X, Boeynaems, Jean-Marie, Barbaras, R, Jacquet, S, Malaval, C, Martinez, Laurent O, Sak, Katrin, Rolland, C, Perez, C, Nauze, M, Champagne, E, Tercé, F, Gachet, Christian, Perret, B, Collet, X, Boeynaems, Jean-Marie, and Barbaras, R
- Abstract
Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein-coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y1 and P2Y13 are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y13 as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y13-mediated HDL endocytosis., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published
- Published
- 2005
4. The nucleotide receptor P2Y13 is a key regulator of hepatic High-Density Lipoprotein (HDL) endocytosis.
- Author
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Jacquet, S., Malaval, C., Martinez, L. O., Sak, K., Rolland, C., Perez, C., Nauze, M., Champagne, E., Tercé, F., Gachet, C., Perret, B., Collet, X., Boeynaems, J.-M., and Barbaras, R.
- Subjects
LIVER cells ,ENDOCYTOSIS ,ABSORPTION (Physiology) ,CELL physiology ,CELL membranes ,ADENOSINE triphosphatase ,PHYSIOLOGICAL control systems ,ISOPENTENOIDS ,LOW-cholesterol diet ,NUCLEIC acids - Abstract
Cell surface receptors for high-density lipoprotein (HDL) on hepatocytes are major partners in the regulation of cholesterol homeostasis. We recently identified a cell surface ATP synthase as a high-affinity receptor for HDL apolipoprotein A-I (apoA-I) on human hepatocytes. Stimulation of this ectopic ATP synthase by apoA-I triggered a low-affinity-receptor-dependent HDL endocytosis by a mechanism strictly related to the generation of ADP. This suggests that nucleotide G-protein- coupled receptors of the P2Y family are molecular components in this pathway. Only P2Y
1 and P2Y13 are present on the membrane of hepatocytes. Using both a pharmacological approach and small interference RNA, we identified P2Y13 as the main partner in hepatic HDL endocytosis, in cultured cells as well as in situ in perfused mouse livers. We also found a new important action of the antithrombotic agent AR-C69931MX as a strong activator of P2Y13 -mediated HDL endocytosis. [ABSTRACT FROM AUTHOR]- Published
- 2005
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5. P316 NEW SPLICING MUTATIONS OF MTP LEADING TO SEVERE ABETALIPOPROTEINEMIA
- Author
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Mas, E., Pons, V., Rolland, C., Nauze, M., Danjoux, M., Gaibelet, G., Sassolas, A., Lévy, E., Tercé, F., and Collet, X.
- Published
- 2010
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6. Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion.
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Delagebeaudeuf, C, Gassama-Diagne, A, Nauze, M, Ragab, A, Li, R Y, Capdevielle, J, Ferrara, P, Fauvel, J, and Chap, H
- Abstract
Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.
- Published
- 1998
7. Mo-W11:6 Accelerated lipid absorption in mice overexpressing intestinal SR-BI
- Author
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Bietrix, F., Daogang, Y., Nauze, M., Rolland, C., Coméra, C., Barbaras, R., Groen, A.K., Perret, B., Tercé, F., and Collet, X.
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- 2006
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8. Étude de l’absorption intestinale du cholestérol sur des souris surexprimant SR-BI au niveau intestinal
- Author
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Bietrix, F., Daogang, Y., Ferrand, C., Rolland, C., Nauze, M., Barbaras, R., Tercé, F., Perret, B., and Collet, X.
- Published
- 2004
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9. Biochemical Characterization and Cloning of Guinea Pig Intestinal Phospholipase B.
- Author
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DELAGEBEAUDEUF, C., GASSAMA-DIAGNE, A., NAUZE, M., RAGAB, A., LI, R. Y., CAPDEVIELLE, J., FERRARA, P., FAUVEL, J., and CHAP, H.
- Published
- 1998
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10. Deciphering biased inverse agonism of cangrelor and ticagrelor at P2Y 12 receptor.
- Author
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Garcia C, Maurel-Ribes A, Nauze M, N'Guyen D, Martinez LO, Payrastre B, Sénard JM, Galés C, and Pons V
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- Adenosine Monophosphate pharmacology, Blotting, Western, Enzyme-Linked Immunosorbent Assay, HEK293 Cells, Humans, Models, Biological, Mutation, Protein Conformation, Protein Stability drug effects, Purinergic P2Y Receptor Agonists pharmacology, Receptors, Cell Surface chemistry, Receptors, Cell Surface metabolism, Receptors, Cell Surface ultrastructure, Receptors, Purinergic P2Y12 chemistry, Receptors, Purinergic P2Y12 genetics, Signal Transduction drug effects, Thrombosis drug therapy, Thrombosis physiopathology, Adenosine Monophosphate analogs & derivatives, Ticagrelor pharmacology
- Abstract
P2Y
12 receptor (P2Y12 -R) is one of the major targets for drug inhibiting platelet aggregation in the treatment/prevention of arterial thrombosis. However, the clinical use of P2Y12 -R antagonists faces some limitations, such as a delayed onset of action (clopidogrel) or adverse effect profile (ticagrelor, cangrelor), justifying the development of a new generation of P2Y12 -R antagonists with a better clinical benefit-risk balance. Although the recent concept of biased agonism offers the possibility to alleviate undesirable adverse effects while preserving therapeutic outcomes, it has never been explored at P2Y12 -R. For the first time, using highly sensitive BRET2-based probes, we accurately delineated biased ligand efficacy at P2Y12 -R in living HEK293T cells on G protein activation and downstream effectors. We demonstrated that P2Y12 -R displayed constitutive Gi/o-dependent signaling that is impaired by the R122C mutation, previously associated with a bleeding disorder. More importantly, we reported the biased inverse agonist efficacy of cangrelor and ticagrelor that could underlie their clinical efficacy. Our study points out that constitutive P2Y12 -R signaling is a normal feature of the receptor that might be essential for platelets to respond faster to a vessel injury. From a therapeutic standpoint, our data suggest that the beneficial advantages of antiplatelet drugs might be more related to inverse agonism at P2Y12 -R than to antagonism of ADP-mediated signaling. In the future, deciphering P2Y12 -R constitutive activity should allow the discovery of more selective biased P2Y12 -R blockers demonstrating therapeutic advantages over classical antiplatelet drugs by improving therapeutic outcomes and concomitantly relieving undesirable adverse effects.- Published
- 2019
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11. Exposure to dietary lipid leads to rapid production of cytosolic lipid droplets near the brush border membrane.
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Soayfane Z, Tercé F, Cantiello M, Robenek H, Nauze M, Bézirard V, Allart S, Payré B, Capilla F, Cartier C, Peres C, Al Saati T, Théodorou V, Nelson DW, Yen CE, Collet X, and Coméra C
- Abstract
Background: Intestinal absorption of dietary lipids involves their hydrolysis in the lumen of proximal intestine as well as uptake, intracellular transport and re-assembly of hydrolyzed lipids in enterocytes, leading to the formation and secretion of the lipoproteins chylomicrons and HDL. In this study, we examined the potential involvement of cytosolic lipid droplets (CLD) whose function in the process of lipid absorption is poorly understood., Methods: Intestinal lipid absorption was studied in mouse after gavage. Three populations of CLD were purified by density ultracentrifugations, as well as the brush border membranes, which were analyzed by western-blots. Immunofluorescent localization of membranes transporters or metabolic enzymes, as well as kinetics of CLD production, were also studied in intestine or Caco-2 cells., Results: We isolated three populations of CLD (ranging from 15 to 1000 nm) which showed differential expression of the major lipid transporters scavenger receptor BI (SR-BI), cluster of differentiation 36 (CD-36), Niemann Pick C-like 1 (NPC1L1), and the ATP-binding cassette transporters ABCG5/G8 but also caveolin 2 and fatty acid binding proteins. The enzyme monoacylglycerol acyltransferase 2 (MGAT2) was identified in the brush border membrane (BBM) in addition to the endoplasmic reticulum, suggesting local synthesis of triglycerides and CLD at both places., Conclusions: We show a very fast production of CLD by enterocytes associated with a transfer of apical constituents as lipid transporters. Our findings suggest that following their uptake by enterocytes, lipids can be partially metabolized at the BBM and packaged into CLD for their transportation to the ER.
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- 2016
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12. Role of the ubiquitin-proteasome system in the regulation of P2Y13 receptor expression: impact on hepatic HDL uptake.
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Pons V, Serhan N, Gayral S, Malaval C, Nauze M, Malet N, Laffargue M, Galés C, and Martinez LO
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- Amino Acid Sequence, Animals, Cell Membrane metabolism, Endocytosis, Endoplasmic Reticulum metabolism, HEK293 Cells, HeLa Cells, Hep G2 Cells, Humans, Mice, Mice, Inbred C57BL, Mice, Knockout, Proteasome Endopeptidase Complex chemistry, Receptors, Purinergic P2 deficiency, Receptors, Purinergic P2 genetics, Receptors, Purinergic P2Y12 genetics, Receptors, Purinergic P2Y12 metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Ubiquitination, Lipoproteins, HDL metabolism, Liver metabolism, Proteasome Endopeptidase Complex metabolism, Receptors, Purinergic P2 metabolism, Ubiquitin metabolism
- Abstract
The protective effect of high density lipoproteins (HDL) against atherosclerosis is mainly attributed to their capacity to transport excess cholesterol from peripheral tissues back to the liver for further elimination into the bile, a process called reverse cholesterol transport (RCT). Recently, the importance of the P2Y13 receptor (P2Y13-R) was highlighted in HDL metabolism since HDL uptake by the liver was decreased in P2Y13-R deficient mice, which translated into impaired RCT. Here, we investigated for the first time the molecular mechanisms regulating cell surface expression of P2Y13-R. When transiently expressed, P2Y13-R was mainly detected in the endoplasmic reticulum (ER) and strongly subjected to proteasome degradation while its homologous P2Y12 receptor (P2Y12-R) was efficiently targeted to the plasma membrane. We observed an inverse correlation between cell surface expression and ubiquitination level of P2Y13-R in the ER, suggesting a close link between ubiquitination of P2Y13-R and its efficient targeting to the plasma membrane. The C-terminus tail exchange between P2Y13-R and P2Y12-R strongly restored plasma membrane expression of P2Y13-R, suggesting the involvement of the intra-cytoplasmic tail of P2Y13-R in expression defect. Accordingly, proteasomal inhibition increased plasma membrane expression of functionally active P2Y13-R in hepatocytes, and consequently stimulated P2Y13-R-mediated HDL endocytosis. Importantly, proteasomal inhibition strongly potentiated HDL hepatic uptake (>200 %) in wild-type but not in P2Y13-R-deficient mice, thus reinforcing the role of P2Y13-R expression in regulating HDL metabolism. Therefore, specific inhibition of the ubiquitin-proteasome system might be a novel powerful HDL therapy to enhance P2Y13-R expression and consequently promote the overall RCT.
- Published
- 2014
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13. Lipidomic and spatio-temporal imaging of fat by mass spectrometry in mice duodenum during lipid digestion.
- Author
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Seyer A, Cantiello M, Bertrand-Michel J, Roques V, Nauze M, Bézirard V, Collet X, Touboul D, Brunelle A, and Coméra C
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- Acetyl-CoA C-Acetyltransferase genetics, Acetyl-CoA C-Acetyltransferase metabolism, Animals, Biological Transport, Carrier Proteins genetics, Carrier Proteins metabolism, Cholesterol metabolism, Chylomicrons metabolism, Duodenum cytology, Enterocytes cytology, Fatty Acids metabolism, Gene Expression, Intestinal Absorption, Lipase genetics, Lipase metabolism, Lipoproteins, HDL metabolism, Mice, Mice, Inbred C57BL, N-Acetylglucosaminyltransferases genetics, N-Acetylglucosaminyltransferases metabolism, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Sterol Esterase genetics, Sterol Esterase metabolism, Sterol O-Acyltransferase genetics, Sterol O-Acyltransferase metabolism, Taurocholic Acid metabolism, Triglycerides metabolism, Sterol O-Acyltransferase 2, Dietary Fats metabolism, Duodenum metabolism, Enterocytes metabolism, Lipid Metabolism
- Abstract
Intestinal absorption of dietary fat is a complex process mediated by enterocytes leading to lipid assembly and secretion of circulating lipoproteins as chylomicrons, vLDL and intestinal HDL (iHDL). Understanding lipid digestion is of importance knowing the correlation between excessive fat absorption and atherosclerosis. By using time-of-flight secondary ion mass spectrometry (TOF-SIMS), we illustrated a spatio-temporal localization of fat in mice duodenum, at different times of digestion after a lipid gavage, for the first time. Fatty acids progressively increased in enterocytes as well as taurocholic acid, secreted by bile and engaged in the entero-hepatic re-absorption cycle. Cytosolic lipid droplets (CLD) from enterocytes were originally purified separating chylomicron-like, intermediate droplets and smaller HDL-like. A lipidomic quantification revealed their contents in triglycerides, free and esterified cholesterol, phosphatidylcholine, sphingomyelin and ceramides but also in free fatty acids, mono- and di-acylglycerols. An acyl-transferase activity was identified and the enzyme monoacylglycerol acyl transferase 2 (MGAT2) was immunodetected in all CLD. The largest droplets was also shown to contain the microsomal triglyceride transfer protein (MTTP), the acyl-coenzyme A-cholesterol acyltransferases (ACAT) 1 and 2, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). This highlights the fact that during the digestion of fats, enterocyte CLD contain some enzymes involved in the different stages of the metabolism of diet fatty acids and cholesterol, in anticipation of the crucial work of endoplasmic reticulum in the process. The data further underlines the dual role of chylomicrons and iHDL in fat digestion which should help to efficiently complement lipid-lowering therapy.
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- 2013
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14. Respective contributions of intestinal Niemann-Pick C1-like 1 and scavenger receptor class B type I to cholesterol and tocopherol uptake: in vivo v. in vitro studies.
- Author
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Reboul E, Soayfane Z, Goncalves A, Cantiello M, Bott R, Nauze M, Tercé F, Collet X, and Coméra C
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- 4-Chloro-7-nitrobenzofurazan metabolism, Absorption, Animals, Azetidines pharmacology, Bile Acids and Salts metabolism, Caco-2 Cells, Cell Membrane metabolism, Cholesterol metabolism, Chromatography, High Pressure Liquid, Cyclopentanes pharmacology, Duodenum metabolism, Ezetimibe, Gene Expression Profiling, Humans, Jejunum metabolism, Lipid Metabolism, Mice, Mice, Inbred C57BL, Micelles, Thiosemicarbazones pharmacology, Time Factors, Vitamin E metabolism, 4-Chloro-7-nitrobenzofurazan analogs & derivatives, Cholesterol analogs & derivatives, Gene Expression Regulation, Membrane Transport Proteins physiology, Scavenger Receptors, Class B physiology, gamma-Tocopherol metabolism
- Abstract
The intestinal absorption of cholesterol and lipid micronutrients such as vitamin E has been shown to share some common pathways. The present study aims to further compare the uptake of cholesterol ([3H]cholesterol v. 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol)) and tocopherol in Caco-2 TC-7 cells and in mouse intestine, with special focus on the respective roles of scavenger receptor class B type I (SR-BI) and Niemann-Pick C1-like 1 (NPC1L1). Conversely to NBD-cholesterol, the uptakes of [3H]cholesterol and tocopherol by Caco-2 cells were impaired by both block lipid transport-1 and ezetimibe, which inhibit SR-BI and NPC1L1, respectively. These inhibitions occurred only when cholesterol or tocopherol was delivered to cells included in micelles that contained biliary acid and at least oleic acid as a lipid. In vivo, after 2 h of digestion in mice, the uptake of the two cholesterol analogues and of tocopherol all showed distinct patterns along the duodenum-jejunum axis. [3H]Cholesterol uptake, which correlated closely to NPC1L1 mRNA expression in wild-type (wt) mice, was strongly inhibited by ezetimibe. Intestinal SR-BI overexpression did not change NPC1L1 expression and led to a significant increase in [3H]cholesterol uptake in the distal jejunum. Conversely, neither ezetimibe treatment nor SR-BI overexpression had an effect on NBD-cholesterol uptake. However, in contrast with SR-BI mRNA expression, tocopherol absorption increased strongly up to the distal jejunum in wt mice where it was specifically inhibited by ezetimibe, and was increased in the proximal intestine of intestinal SR-BI-overexpressing mice. Thus, cholesterol and tocopherol uptakes share common pathways in cell culture models, but display different in vivo absorption patterns associated with distinct contributions of SR-BI and NPC1L1.
- Published
- 2012
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15. A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP).
- Author
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Pons V, Rolland C, Nauze M, Danjoux M, Gaibelet G, Durandy A, Sassolas A, Lévy E, Tercé F, Collet X, and Mas E
- Subjects
- Agammaglobulinemia genetics, Alternative Splicing genetics, Amino Acid Sequence, Carrier Proteins metabolism, Child, Endoplasmic Reticulum metabolism, Female, HeLa Cells, Hep G2 Cells, Humans, Infant, Male, Microsomes metabolism, Molecular Sequence Data, Mutation genetics, Protein Binding genetics, Protein Disulfide-Isomerases genetics, Protein Disulfide-Isomerases metabolism, Triglycerides metabolism, Abetalipoproteinemia genetics, Abetalipoproteinemia pathology, Carrier Proteins genetics, Exons genetics
- Abstract
Abetalipoproteinemia is a rare autosomal recessive disease characterized by low lipid levels and by the absence of apoB-containing lipoproteins. It is the consequence of microsomal triglyceride transfer protein (MTTP) deficiency. We report two patients with new MTTP mutations. We studied their functional consequences on the triglyceride transfer function using duodenal biopsies. We transfected MTTP mutants in HepG2 and HeLa cells to investigate their association with protein disulfide isomerase (PDI) and their localization at the endoplasmic reticulum. These children have a severe abetalipoproteinemia. Both of them had also a mild hypogammaglobulinemia. They are compound heterozygotes with c.619G>T and c.1237-28A>G mutations within the MTTP gene. mRNA analysis revealed abnormal splicing with deletion of exon 6 and 10, respectively. Deletion of exon 6 (Δ6-MTTP) introduced a frame shift in the reading frame and a premature stop codon at position 234. Despite the fact that Δ6-MTTP and Δ10-MTTP mutants were not capable of binding PDI, both MTTP mutant proteins normally localize at the endoplasmic reticulum. However, these two mutations induce a loss of MTTP triglyceride transfer activity. These two mutations lead to abnormal truncated MTTP proteins, incapable of binding PDI and responsible for the loss of function of MTTP, thereby explaining the severe abetalipoproteinemia phenotype of these children., (© 2011 Wiley-Liss, Inc.)
- Published
- 2011
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16. Accelerated lipid absorption in mice overexpressing intestinal SR-BI.
- Author
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Bietrix F, Yan D, Nauze M, Rolland C, Bertrand-Michel J, Coméra C, Schaak S, Barbaras R, Groen AK, Perret B, Tercé F, and Collet X
- Subjects
- Absorption, Animals, Apolipoproteins chemistry, Bile Acids and Salts metabolism, Cell Membrane metabolism, Cholesterol chemistry, Cholesterol metabolism, Chylomicrons chemistry, DNA, Complementary metabolism, Homeostasis, Immunohistochemistry, Intestines chemistry, Lipoproteins chemistry, Liver metabolism, Mice, Mice, Transgenic, Promoter Regions, Genetic, Receptors, Lipoprotein metabolism, Receptors, Scavenger chemistry, Tissue Distribution, Triglycerides metabolism, Triolein chemistry, Intestinal Mucosa metabolism, Lipids chemistry, Scavenger Receptors, Class B metabolism
- Abstract
Dietary cholesterol absorption contributes to a large part of the circulating cholesterol. However, the mechanism of sterol intestinal uptake is not clearly elucidated. Scavenger receptor class B type I (SR-BI), major component in the control of cholesterol homeostasis, is expressed in the intestine, but its role in this organ remains unclear. We have generated transgenic mice overexpressing SR-BI primarily in the intestine by using the mouse SR-BI gene under the control of intestinal specific "apoC-III enhancer coupled with apoA-IV promoter." We found SR-BI overexpression with respect to the natural protein along the intestine and at the top of the villosities. After a meal containing [(14)C]cholesterol and [(3)H]triolein, SR-BI transgenic mice presented a rise in intestinal absorption of both lipids that was not due to a defect in chylomicron clearance nor to a change in the bile flow or the bile acid content. Nevertheless, SR-BI transgenic mice showed a decrease of total cholesterol but an increase of triglyceride content in plasma without any change in the high density lipoprotein apoA-I level. Thus, we described for the first time a functional role in vivo for SR-BI in cholesterol but also in triglyceride intestinal absorption.
- Published
- 2006
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17. Enterophilin-1 interacts with focal adhesion kinase and decreases beta1 integrins in intestinal Caco-2 cells.
- Author
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Pons V, Pérès C, Teulié JM, Nauze M, Mus M, Rolland C, Collet X, Perret B, Gassama-Diagne A, and Hullin-Matsuda F
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- Caco-2 Cells, Carrier Proteins biosynthesis, Carrier Proteins genetics, Cell Differentiation genetics, Down-Regulation, Focal Adhesion Kinase 1, Focal Adhesion Protein-Tyrosine Kinases, Gene Expression Regulation, HeLa Cells, Humans, Integrin beta1 genetics, Intestinal Mucosa metabolism, Intestinal Mucosa pathology, Carrier Proteins metabolism, Integrin beta1 biosynthesis, Protein-Tyrosine Kinases metabolism, Vesicular Transport Proteins
- Abstract
Intestinal cell growth and differentiation are tightly regulated by growth factors and extracellular matrix components along the crypt-villus axis. We previously described enterophilin-1 (Ent-1) as a new intestinal protein associated with growth arrest and enterocyte differentiation. Ent-1 interacted with sorting nexin 1 and decreased cell surface epidermal growth factor receptor. Because beta(1) integrins are mostly found in vivo in the proliferative crypt cells, we investigated the role of Ent-1 in the fate of beta(1) integrin subunits. In undifferentiated intestinal Caco-2 cells, overexpression of Ent-1 induces a marked decrease of alpha(5)beta(1) integrin pools, whereas alpha(2)beta(1) integrin is weakly affected. Conversely, overexpression of sorting nexin 1 has no effect on integrin levels despite its ability to interact with Ent-1. Interestingly, we identified focal adhesion kinase as a new Ent-1 partner using yeast two-hybrid screening and co-precipitation experiments. Furthermore by confocal microscopy, we observed that Ent-1 and beta(1) integrins partly co-localize on vesicular structures, suggesting a role for Ent-1 in integrin trafficking. Because focal adhesion kinase is able to bind both Ent-1 and beta(1) integrins, the kinase might act as a molecular bridge between the two proteins. Altogether, these results support a role of Ent-1 in regulating beta(1) integrin expression that could favor intestinal differentiation.
- Published
- 2004
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18. Enterophilin-1, a new partner of sorting nexin 1, decreases cell surface epidermal growth factor receptor.
- Author
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Pons V, Hullin-Matsuda F, Nauze M, Barbaras R, Pérès C, Collet X, Perret B, Chap H, and Gassama-Diagne A
- Subjects
- Animals, COS Cells, Caco-2 Cells, Carrier Proteins genetics, Cell Differentiation, Endosomes metabolism, Enterocytes cytology, Enterocytes metabolism, Gene Library, HeLa Cells, Humans, Kidney cytology, Lysosomes metabolism, Macromolecular Substances, Membrane Proteins metabolism, Protein Transport physiology, Two-Hybrid System Techniques, Carrier Proteins metabolism, Endocytosis physiology, ErbB Receptors metabolism, Vesicular Transport Proteins
- Abstract
We previously described enterophilin-1 (Ent-1), a new intestinal protein bearing an extended leucine zipper and a B30.2 domain. Ent-1 expression is associated with growth arrest and enterocyte differentiation. To investigate the importance of Ent-1 in the differentiation, we performed a yeast two-hybrid screening. We identified sorting nexin 1 (SNX1) as a novel partner of Ent-1 and confirmed the specificity of interaction by co-immunoprecipitation experiments in mammalian cells. SNX1 is associated with endosomal membranes and triggers the endosome-to-lysosome pathway of epidermal growth factor receptor (EGFR). We observe by immunofluorescence microscopy that Ent-1 and SNX1 are co-localized on vesicular and tubulovesicular structures, which are different from early endosome antigen 1-containing endosomes. By gel filtration chromatography, we show that Ent-1 and SNX1 co-eluted in macromolecular complexes containing part of EGFR. Furthermore, overexpressed Ent-1 decreases cell surface EGFR. Ent-1 and SNX1 co-overexpression strongly extends EGFR diminution, indicating a synergetic effect of both proteins on cell surface EGFR removal. Interestingly, the increase of endogenous Ent-1 expression correlates with the decrease of EGFR during spontaneous differentiation of Caco-2 cells. We thus propose a role of Ent-1 in the trafficking of EGFR to down-regulate intestinal mitogenic signals, highlighting the mechanisms of cell growth arrest associated with enterocytic differentiation.
- Published
- 2003
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19. Guinea pig phospholipase B, identification of the catalytic serine and the proregion involved in its processing and enzymatic activity.
- Author
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Nauze M, Gonin L, Chaminade B, Perès C, Hullin-Matsuda F, Perret B, Chap H, and Gassama-Diagne A
- Subjects
- Animals, Binding Sites, COS Cells, Catalytic Domain, Cell Membrane metabolism, Gene Deletion, Glycoside Hydrolases metabolism, Glycosylation, Guinea Pigs, Immunoblotting, Kinetics, Microscopy, Confocal, Microvilli metabolism, Mutagenesis, Site-Directed, Mutation, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins metabolism, Serine metabolism, Time Factors, Transfection, Trypsin metabolism, Lysophospholipase chemistry, Lysophospholipase metabolism, Serine chemistry
- Abstract
Guinea pig phospholipase B (GPPLB) is a glycosylated ectoenzyme of intestinal brush border membrane. It displays a broad substrate specificity and is activated by trypsin cleavage. The primary sequence contains four tandem repeat domains (I to IV) and several serines in lipase consensus sequences. We used site-directed mutagenesis to demonstrate that only the serine 399 present in repeat II is responsible for the various enzymatic activities of GPPLB. Furthermore, we characterized for the first time the retinyl esterase activity of the enzyme. We also constructed and expressed in COS-7 cells, an NH(2)-terminal repeat I deletion mutant which was detected at a very low level by immunoblot. However, confocal microscopy study showed a strong intracellular accumulation with a weak membrane expression of the mutated protein, indicating a role of the NH(2)-terminal repeat I in the processing of GPPLB. Nevertheless, the Western blot-detected protein presented a glycosylation and trypsin sensitivity patterns similar to wild type PLB. The mutant is also fully active without trypsin treatment, in contrast to native enzyme. Thus, we propose a structural model for GPPLB, in which the repeat I constitutes a lid covering the active site and impairing enzymatic activity, its removal by trypsin leading to an active protein.
- Published
- 2002
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20. Human epidermis is a novel site of phospholipase B expression.
- Author
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Maury E, Prévost MC, Nauze M, Redoulès D, Tarroux R, Charvéron M, Salles JP, Perret B, Chap H, and Gassama-Diagne A
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, DNA Probes, Fatty Acids, Nonesterified biosynthesis, Humans, In Situ Hybridization, Lysophospholipase chemistry, Lysophospholipase genetics, Molecular Sequence Data, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Subcellular Fractions enzymology, Epidermis enzymology, Lysophospholipase metabolism
- Abstract
Phospholipase B (PLB) is an enzyme that displays both phospholipase A(2) and lysophospholipase activities. Analysis of human epidermis homogenates indicated the presence of a 97 kDa PLB protein, as well as a phospholipase A(2) activity, both being enriched in the soluble fraction. Immunolabelling and in situ hybridization experiments showed that this enzyme is expressed in the different layers of epidermis with an accumulation at the dermo-epidermis junction. RT-PCR data indicated that PLB is specifically expressed in natural and reconstructed epidermis. By 3'-RACE-PCR and screening of human genome databases, we obtained a 3600 bp cDNA coding for human PLB highly homologous to already described intestinal brush border PLBs. These data led us to conclude that the soluble PLB corresponds to a proteolytic cleavage of the membrane anchored protein. Altogether, our results provide the first characterization of human PLB which should play an important role in epidermal barrier function.
- Published
- 2002
- Full Text
- View/download PDF
21. Enterophilins, a new family of leucine zipper proteins bearing a b30.2 domain and associated with enterocyte differentiation.
- Author
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Gassama-Diagne A, Hullin-Matsuda F, Li RY, Nauze M, Ragab A, Pons V, Delagebeaudeuf C, Simon MF, Fauvel J, and Chap H
- Subjects
- Amino Acid Sequence, Animals, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Differentiation physiology, Cells, Cultured, Cloning, Molecular, DNA, Complementary analysis, DNA, Complementary genetics, Enterocytes cytology, Molecular Sequence Data, Proteins metabolism, Sequence Alignment, Enterocytes physiology, Leucine Zippers, Proteins genetics
- Abstract
Enterocyte terminal differentiation occurs at the crypt-villus junction through the transcriptional activation of cell-specific genes, many of which code for proteins of the brush border membrane such as intestinal alkaline phosphatase, sucrase-isomaltase, or the microvillar structural protein villin. Several studies have shown that this sharp increase in specific mRNA levels is intimately associated with arrest of cell proliferation. We isolated several clones from a guinea pig intestine cDNA library. They encode new proteins characterized by an original structure associating a carboxyl-terminal B30.2/RFP-like domain and a long leucine zipper at the amino terminus. The first member of this novel gene family codes for a 65-kDa protein termed enterophilin-1, which is specifically expressed in enterocytes before their final differentiation. Enterophilin-1 is the most abundant in the small intestine but is still present in significant amounts in colonic enterocytes. In Caco-2 cells, a similar 65-kDa protein was recognized by a specific anti-enterophilin-1 antibody, and its expression was positively correlated with cell differentiation status. In addition, transfection of HT-29 cells with enterophilin-1 full-length cDNA slightly inhibited cell growth and promoted an increase in alkaline phosphatase activity. Taken together, these data identify enterophilins as a new family of proteins associated with enterocyte differentiation.
- Published
- 2001
- Full Text
- View/download PDF
22. New developments in phospholipase A2.
- Author
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Chaminade B, Le Balle F, Fourcade O, Nauze M, Delagebeaudeuf C, Gassama-Diagne A, Simon MF, Fauvel J, and Chap H
- Subjects
- Animals, Calcium metabolism, Cell Membrane enzymology, Cytosol enzymology, Humans, Lysophospholipase chemistry, Lysophospholipase genetics, Phospholipases A2, Recombinant Proteins metabolism, Phospholipases A chemistry, Phospholipases A metabolism
- Abstract
Some of the most recent data concerning various phospholipases A2, with special emphasis on secretory, cytosolic, and calcium-independent phospholipases A2 are summarized. Besides their contribution to the production of proinflammatory lipid mediators, the involvement of these enzymes in key cell responses such as apoptosis or tumor cell metastatic potential is also discussed, taking advantage of transgenic models based on gene invalidation by homologous recombination. The possible role of secretory and cytosolic platelet-activating factor acetyl hydrolases is also briefly mentioned. Finally, the ectopic expression in epididymis of an intestinal phospholipase B opens some novel issues as to the possible function of phospholipases in reproduction.
- Published
- 1999
- Full Text
- View/download PDF
23. Guinea pig intestinal phospholipase B: protein expression during enterocyte maturation and effects of N-oligosaccharide removal on enzymatic activities and protein stability.
- Author
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Delagebeaudeuf C, Gassama A, Collet X, Nauze M, and Chap H
- Subjects
- Animals, Cell Differentiation, Enzyme Induction, Glycoside Hydrolases pharmacology, Glycosylation, Intestine, Small ultrastructure, Kinetics, Lectins metabolism, Lysophospholipase chemistry, Lysophospholipase drug effects, Lysophospholipase genetics, Microvilli enzymology, Molecular Weight, Protein Denaturation, Guinea Pigs metabolism, Intestine, Small enzymology, Lysophospholipase biosynthesis, Oligosaccharides chemistry
- Abstract
Guinea pig phospholipase B (PLB) is an intestinal brush-border hydrolase displaying a broad substrate specificity towards various dietary lipids. PLB was detected by immunoblotting as a single 140-kDa polypeptide in all cell populations isolated from guinea pig intestinal mucosa, but increased in parallel to its activity from undifferentiated to mature cells, the specific activity of the enzyme remaining constant. Moreover, N-glycosylation, which contributed to 23% of the apparent molecular mass, was identical along the cell differentiation axis. In all cell fractions, N-linked sugar chains were of the complex type, since they were removed by N-glycosidase F, whereas PLB remained insensitive to endoglycosidase H. Moreover, lack of O-glycosylation was demonstrated by the insensitivity of PLB to O-glycosidase and by its failure to interact with Helix pomatia lectin after prior treatment with neuraminidase or alpha-fucosidase. Enzymatic removal of sugar chains reduced phospholipase A2, lysophospholipase and diacylglycerol lipase activities by 27-35%, kinetic analysis indicating a decrease in apparent Vmax values for the three enzymatic activities, whereas the Km remained unchanged. Finally, the carbohydrate-depleted form of PLB did not display gross changes in thermal stability, in contrast to PLB from microorganisms previously investigated. Our data indicate that the high level of PLB N-glycosylation is poorly related to its biological function. Whether carbohydrate chains are involved in proper targeting of the enzyme to the brush-border membrane remains to be established.
- Published
- 1996
- Full Text
- View/download PDF
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