RESUMO
P2Y12 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.
Assuntos
Monofosfato de Adenosina/análogos & derivados , Ticagrelor/farmacologia , Monofosfato de Adenosina/farmacologia , Western Blotting , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Modelos Biológicos , Mutação , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2Y/farmacologia , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/ultraestrutura , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/genética , Transdução de Sinais/efeitos dos fármacos , Trombose/tratamento farmacológico , Trombose/fisiopatologiaRESUMO
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.
Assuntos
Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Purinérgicos P2/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Endocitose , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexo de Endopeptidases do Proteassoma/química , Receptores Purinérgicos P2/deficiência , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2Y12/genética , Receptores Purinérgicos P2Y12/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , UbiquitinaçãoRESUMO
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.
Assuntos
4-Cloro-7-nitrobenzofurazano/análogos & derivados , Colesterol/análogos & derivados , Regulação da Expressão Gênica , Proteínas de Membrana Transportadoras/fisiologia , Receptores Depuradores Classe B/fisiologia , gama-Tocoferol/metabolismo , 4-Cloro-7-nitrobenzofurazano/metabolismo , Absorção , Animais , Azetidinas/farmacologia , Ácidos e Sais Biliares/metabolismo , Células CACO-2 , Membrana Celular/metabolismo , Colesterol/metabolismo , Cromatografia Líquida de Alta Pressão , Ciclopentanos/farmacologia , Duodeno/metabolismo , Ezetimiba , Perfilação da Expressão Gênica , Humanos , Jejuno/metabolismo , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos C57BL , Micelas , Tiossemicarbazonas/farmacologia , Fatores de Tempo , Vitamina E/metabolismoRESUMO
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.
Assuntos
Abetalipoproteinemia/genética , Abetalipoproteinemia/patologia , Proteínas de Transporte/genética , Éxons/genética , Agamaglobulinemia/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Criança , Retículo Endoplasmático/metabolismo , Feminino , Células HeLa , Células Hep G2 , Humanos , Lactente , Masculino , Microssomos/metabolismo , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica/genética , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Triglicerídeos/metabolismoRESUMO
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.
RESUMO
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.
Assuntos
Gorduras na Dieta/metabolismo , Duodeno/metabolismo , Enterócitos/metabolismo , Metabolismo dos Lipídeos , Acetil-CoA C-Acetiltransferase/genética , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Quilomícrons/metabolismo , Duodeno/citologia , Enterócitos/citologia , Ácidos Graxos/metabolismo , Expressão Gênica , Absorção Intestinal , Lipase/genética , Lipase/metabolismo , Lipoproteínas HDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esterol Esterase/genética , Esterol Esterase/metabolismo , Esterol O-Aciltransferase/genética , Esterol O-Aciltransferase/metabolismo , Ácido Taurocólico/metabolismo , Triglicerídeos/metabolismo , Esterol O-Aciltransferase 2RESUMO
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.
Assuntos
Mucosa Intestinal/metabolismo , Lipídeos/química , Receptores Depuradores Classe B/metabolismo , Absorção , Animais , Apolipoproteínas/química , Ácidos e Sais Biliares/metabolismo , Membrana Celular/metabolismo , Colesterol/química , Colesterol/metabolismo , Quilomícrons/química , DNA Complementar/metabolismo , Homeostase , Imuno-Histoquímica , Intestinos/química , Lipoproteínas/química , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Receptores de Lipoproteínas/metabolismo , Receptores Depuradores/química , Distribuição Tecidual , Triglicerídeos/metabolismo , Trioleína/químicaRESUMO
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.
Assuntos
Lisofosfolipase/química , Lisofosfolipase/metabolismo , Serina/química , Animais , Sítios de Ligação , Células COS , Domínio Catalítico , Membrana Celular/metabolismo , Deleção de Genes , Glicosídeo Hidrolases/metabolismo , Glicosilação , Cobaias , Immunoblotting , Cinética , Microscopia Confocal , Microvilosidades/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Fatores de Tempo , Transfecção , Tripsina/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte/metabolismo , Integrina beta1/biossíntese , Proteínas Tirosina Quinases/metabolismo , Proteínas de Transporte Vesicular , Células CACO-2 , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Diferenciação Celular/genética , Regulação para Baixo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Regulação da Expressão Gênica , Células HeLa , Humanos , Integrina beta1/genética , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologiaRESUMO
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.
Assuntos
Epiderme/enzimologia , Lisofosfolipase/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sondas de DNA , Ácidos Graxos não Esterificados/biossíntese , Humanos , Hibridização In Situ , Lisofosfolipase/química , Lisofosfolipase/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Frações Subcelulares/enzimologiaRESUMO
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.