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ABSTRACT: MicroRNAs are small non-coding RNAs that physiologically modulate proteins expression, and regulate numerous cellular mechanisms. Alteration of microRNA expression has been described in cancer and is associated to tumor initiation and progression. The microRNA 148a (miR-148a) is frequently down-regulated in cancer. We previously demonstrated that its down-regulation by DNA hypermethylation is an early event in pancreatic ductal adenocarcinoma (PDAC) carcinogenesis, suggesting a tumor suppressive function. Here, we investigate the potential role of miR-148a over-expression in PDAC as a therapeutic tool. We first report the consequences of miR-148a over-expression in PDAC cell lines. We demonstrate that miR-148a over-expression has no dramatic effect on cell proliferation and cell chemo-sensitivity in four well described PDAC cell lines. We also investigate the modulation of protein expression by a global proteomic approach (2D-DIGE). We show that despite its massive over-expression, miR-148a weakly modulates protein expression, thus preventing the identification of protein targets in PDAC cell lines. More importantly, in vivo data demonstrate that modulating miR-148a expression either in the epithelia tumor cells and/or in the tumor microenvironment does not impede tumor growth. Taken together, we demonstrate herein that miR-148a does not impact PDAC proliferation both in vitro and in vivo thus suggesting a weak potential as a therapeutic tool.
PLoS ONE 01/2013; 8(1):e55513. · 4.09 Impact Factor
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Annelise Genoux,
Véronique Pons,
Claudia Radojkovic,
Florence Roux-Dalvai,
Guillaume Combes,
Corinne Rolland,
Nicole Malet,
Bernard Monsarrat, Frédéric Lopez,
Jean-Bernard Ruidavets,
Bertrand Perret,
Laurent O Martinez
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ABSTRACT: Mitochondrial ATP synthase is expressed as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein component in High Density Lipoproteins (HDL). On hepatocytes, apoA-I binds to cell surface ATP synthase (namely ecto-F(1)-ATPase) and stimulates its ATPase activity, generating extracellular ADP. This production of extracellular ADP activates a P2Y(13)-mediated HDL endocytosis pathway. Conversely, exogenous IF1, classically known as a natural mitochondrial specific inhibitor of F(1)-ATPase activity, inhibits ecto-F(1)-ATPase activity and decreases HDL endocytosis by both human hepatocytes and perfused rat liver.
Since recent reports also described the presence of IF1 at the plasma membrane of different cell types, we investigated whether IF1 is present in the systemic circulation in humans. We first unambiguously detected IF1 in human serum by immunoprecipitation and mass spectrometry. We then set up a competitive ELISA assay in order to quantify its level in human serum. Analyses of IF1 levels in 100 normolipemic male subjects evidenced a normal distribution, with a median value of 0.49 µg/mL and a 95% confidence interval of 0.22-0.82 µg/mL. Correlations between IF1 levels and serum lipid levels demonstrated that serum IF1 levels are positively correlated with HDL-cholesterol and negatively with triglycerides (TG).
Altogether, these data support the view that, in humans, circulating IF1 might affect HDL levels by inhibiting hepatic HDL uptake and also impact TG metabolism.
PLoS ONE 01/2011; 6(9):e23949. · 4.09 Impact Factor
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ABSTRACT: Juvenile hormone (JH) controls insect development, metamorphosis and reproduction. In insect hemolymph a significant proportion of JH is bound to juvenile hormone binding protein (JHBP), which serves as a carrier supplying the hormone to the target tissues. To shed some light on JHBP passage within insect tissues, the interaction of this carrier with other proteins from Galleria mellonella (Lepidoptera) was investigated. Our studies revealed the presence of JHBP within the tracheal epithelium and fat body cells in both the membrane and cytoplasmic sections. We found that the interaction between JHBP and membrane proteins occurs with saturation kinetics and is specific and reversible. ATP synthase was indicated as a JHBP membrane binding protein based upon SPR-BIA and MS analysis. It was found that in G. mellonella fat body, this enzyme is present in mitochondrial fraction, plasma membranes and cytosol as well. In the model system containing bovine F(1) ATP synthase and JHBP, the interaction between these two components occurs with K(d)=0.86 nM. In hemolymph we detected JHBP binding to apolipophorin, arylphorin and hexamerin. These results provide the first demonstration of the physical interaction of JHBP with membrane and hemolymph proteins which can be involved in JHBP molecule traffic.
Biochimica et Biophysica Acta 06/2009; 1788(9):1695-705. · 4.66 Impact Factor
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ABSTRACT: Somatostatin is a neuropeptide that inhibits exocrine and endocrine secretions of several hormones and negatively regulates cell proliferation. These events are mediated through somatostatin engagement on one of five G protein-coupled receptors named SSTR1 to STTR5. Somatostatin binding to SSTR2 mediates predominantly antisecretory and antiproliferative effects; two important biological activities in the gastroenteropancreatic endocrine and exocrine system. Herein we demonstrate novel regulatory sequences for human (h) SSTR2 transcription. By genomic DNA sequence analysis, we reveal two CpG islands located 3.8 kb upstream from the transcription start site. We identify a novel transcription start site and a promoter region within one of these CpG islands. We demonstrate that two epigenetic modifications, DNA methylation and histone acetylation, regulate the activation of hSSTR2 upstream promoter. Furthermore, we show that the transcription from this upstream promoter region directly correlates to hSSTR2 mRNA expression in various human cell lines. A combined treatment of a demethylating agent, 5-aza-2-deoxycytidine and a histone deacetylase inhibitor, trichostatin A, leads to increased expression of hSSTR2 mRNA in cell lines in which the CpG island is methylated. The epigenetic regulation of this promoter region results in differential expression of hSSTR2 mRNA in human cell lines. This study reveals the existence of a novel upstream promoter for the hSSTR2 gene that is regulated by epigenetic modifications, suggesting for complex control of the hSSTR2 transcription.
Endocrinology 07/2008; 149(6):3137-47. · 4.46 Impact Factor
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Journal of the American Chemical Society 03/2007; 129(6):1502-3. · 9.91 Impact Factor
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Corinne Bousquet,
Julie Guillermet-Guibert,
Nathalie Saint-Laurent,
Elodie Archer-Lahlou, Frédéric Lopez,
Marjorie Fanjul,
Audrey Ferrand,
Daniel Fourmy,
Carole Pichereaux,
Bernard Monsarrat,
Lucien Pradayrol,
Jean-Pierre Estève,
Christiane Susini
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ABSTRACT: Phosphatidylinositol 3-kinase (PI3K) regulates many cellular functions including growth and survival, and its excessive activation is a hallmark of cancer. Somatostatin, acting through its G protein-coupled receptor (GPCR) sst2, has potent proapoptotic and anti-invasive activities on normal and cancer cells. Here, we report a novel mechanism for inhibiting PI3K activity. Somatostatin, acting through sst2, inhibits PI3K activity by disrupting a pre-existing complex comprising the sst2 receptor and the p85 PI3K regulatory subunit. Surface plasmon resonance and molecular modeling identified the phosphorylated-Y71 residue of a p85-binding pYXXM motif in the first sst2 intracellular loop, and p85 COOH-terminal SH2 as direct interacting domains. Somatostatin-mediated dissociation of this complex as well as p85 tyrosine dephosphorylation correlates with sst2 tyrosine dephosphorylation on the Y71 residue. Mutating sst2-Y71 disabled sst2 to interact with p85 and somatostatin to inhibit PI3K, consequently abrogating sst2's ability to suppress cell survival and tumor growth. These results provide the first demonstration of a physical interaction between a GPCR and p85, revealing a novel mechanism for negative regulation by ligand-activated GPCR of PI3K-dependent survival pathways, which may be an important molecular target for antineoplastic therapy.
The EMBO Journal 10/2006; 25(17):3943-54. · 9.20 Impact Factor
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ABSTRACT: Recently, we have described a novel protein-protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)gamma1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCgamma1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCgamma1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCgamma1. Finally we also identified bradykinin-induced PLCgamma1 recruitment and activation in primary culture renal mesangial cells.
Biochemical and Biophysical Research Communications 02/2005; 326(4):894-900. · 2.48 Impact Factor
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ABSTRACT: The capacity of G-quadruplex ligands to stabilize four-stranded DNA makes them able to inhibit telomerase, which is involved in tumour cell proliferation. A series of cationic metalloporphyrin derivatives was prepared by making variations on a meso-tetrakis(4-N-methyl-pyridiniumyl)porphyrin skeleton (TMPyP). The DNA binding properties of nickel(II) and manganese(III) porphyrins were studied by surface plasmon resonance, and the capacity of the nickel porphyrins to inhibit telomerase was tested in a TRAP assay. The nature of the metal influences the kinetics (the process is faster for Ni than for Mn) and the mode of interaction (stacking or external binding). The chemical alterations did not lead to increased telomerase inhibition. The best selectivity for G-quadruplex DNA was observed for Mn-TMPyP, which has a tenfold preference for quadruplex over duplex.
ChemBioChem 02/2005; 6(1):123-32. · 3.94 Impact Factor
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Emmanuel Scotet,
Laurent O Martinez,
Ethan Grant,
Ronald Barbaras,
Paul Jenö,
Martine Guiraud,
Bernard Monsarrat,
Xavier Saulquin,
Sophie Maillet,
Jean-Pierre Estève, Frédéric Lopez,
Bertrand Perret,
Xavier Collet,
Marc Bonneville,
Eric Champagne
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ABSTRACT: 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.
Immunity 02/2005; 22(1):71-80. · 21.64 Impact Factor
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Emmanuel Scotet,
Laurent O. Martinez,
Ethan Grant,
Ronald Barbaras,
Paul Jenö,
Martine Guiraud,
Bernard Monsarrat,
Xavier Saulquin,
Sophie Maillet,
Jean-Pierre Estève, Frédéric Lopez,
Bertrand Perret
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ABSTRACT: Vγ9Vδ2 T lymphocytes, a major γδ 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 Vγ9Vδ2 T cells. When immobilized, purified F1-ATPase induces selective activation of this lymphocyte subset. The Vγ9Vδ2 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 Vγ9Vδ2 T cells by tumors expressing F1-ATPase. This study thus describes an unanticipated tumor recognition mechanism by Vγ9Vδ2 lymphocytes and a possible link between γδ T cell immunity and lipid metabolism.
Immunity 01/2005; 22(1):71-80. · 21.64 Impact Factor
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ABSTRACT: Corneodesmosin (CDSN), a glycoprotein expressed during the late stages of epidermal differentiation, localizes in the extracellular core of upper desmosomes and of corneodesmosomes. Since it displays homophilic adhesive properties, CDSN is thought to reinforce cell-cell cohesion within the upper layers of the epidermis. CDSN presents two serine- and glycine-rich domains in its N- and C-terminus that may fold into highly flexible and adhesive secondary structures called glycine loops. We analyzed the importance of these domains in CDSN homophilic adhesion by producing full-length and truncated recombinant forms of the protein deleted of the N- and/or the C-terminal domain. The adhesive properties of the various proteins were then tested in vitro by overlay binding assays and surface plasmon resonance quantitative analysis. Experiments evidenced the homophilic adhesive properties of the N-terminal glycine loop domain, confirming its involvement in CDSN-CDSN interactions. They further indicated that most of the C-terminal domain is not necessary for the adhesive properties of the protein. The dissociation constant (K(D)) was calculated to be 1.3x10(-5) M. This interaction strength might allow dynamic regulation of the CDSN-CDSN association to occur in vivo. Moreover, molecular filtration analyses demonstrated for the first time that non-glycosylated CDSN is able to spontaneously form large homo-oligomers in vitro and that the N-terminal glycine loop domain is necessary for the formation of these macromolecular complexes.
Journal of Investigative Dermatology 04/2004; 122(3):747-54. · 6.31 Impact Factor
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ABSTRACT: Biological functions of most macromolecules depend on their ability to interact with other molecules and a great challenge is the complete description of the protein interaction networks. Biomolecular interaction analysis (BIA) is an optical technology that uses the surface plasmon resonance phenomenon for characterizing macromolecular interactions between an analyte in solution and its ligand immobilized on a sensor chip. Further identification of interacting proteins can be achieved by combining this nondestructive method to mass spectrometry (MS). The BIA-MS approach represents a promising tool in proteomics for the characterization of protein/protein interactions. In this study, we report on the improved sensitivity in the identification of an unknown protein bound to a known ligand by a rapid and simple BIA-MS approach. We took advantage of a new automatic and very reproducible microelution procedure available on BIACORE 3000 instruments, called "microrecovery", to elute the bound protein from the sensor chip. Protein identification was then achieved after tryptic digestion by matrix-assisted laser desorption/ionization-time of flight mass mapping and database search. The strategy was succesfully applied to the model protein SHP2 tyrosine phosphatase interacting with an immunoreceptor tyrosine-based inhibitory motif sequence of the sst2 somatostatin receptor. Optimization of the BIA-MS approach allowed the unambiguous identification of 10-20 fmol of the protein specifically trapped from a complex mixture of cytosolic extracts.
PROTEOMICS 05/2003; 3(4):402-12. · 4.51 Impact Factor
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ABSTRACT: Recently, we have described a novel protein–protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)γ1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCγ1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCγ1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCγ1. Finally we also identified bradykinin-induced PLCγ1 recruitment and activation in primary culture renal mesangial cells.
Biochemical and Biophysical Research Communications.
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ABSTRACT: Immature porcine Sertoli cells have been reported to be tar- gets for the regulatory peptide somatostatin (SRIF), which inhibits the basal and FSH-induced proliferation of Sertoli cells through a decrease of cAMP production. In the present study, we show that SRIF inhibits both basal and FSH-stimulated expression of the stem cell factor (SCF), a Sertoli cell-specific gene. The SRIF-me- diated inhibition of forskolin-triggered, but not of 8-bromoaden- osine-cAMP-triggered, SCF mRNA expression demonstrates the involvement of adenylyl cyclase in underlying peptide actions. Moreover, these effects require functional coupling of specific plasma membrane receptors to adenylyl cyclase via inhibitory G proteins, because pertussis toxin prevents SRIF-mediated inhibi- tion of SCF mRNA expression. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays suggest the in- volvement of sst2 receptors in SRIF actions on Sertoli cells. The biological relevance of these data is supported by an SRIF-medi- ated decrease in SCF-induced incorporation of ( 3 H)thymidine in isolated seminiferous tubules. In situ hybridization and confocal microscopy show that, in seminiferous tubules only, spermato- gonia display both c-kit and sst2 receptors. Taken together, these results suggest that SCF-stimulated DNA synthesis can be inhib- ited by SRIF in spermatogonia, but not in Sertoli and peritubular cells. Combined RT-PCR and immunohistochemical approaches point toward spermatogonia and Leydig cells as the source of testicular SRIF. These data argue in favor of paracrine/autocrine SRIF actions in testis. cAMP, cytokines, gene regulation, Leydig cells, Sertoli cells, sig- nal transduction, somatostatin
