Pascual Ferrara - Academia.edu (original) (raw)
Papers by Pascual Ferrara
Neuroscience, 1998
Neurons and glial cells are capable of synthesizing various bioactive steroids, but the neuronal ... more Neurons and glial cells are capable of synthesizing various bioactive steroids, but the neuronal mechanisms controlling neurosteroid-secreting cells are poorly understood. In the present study, we have investigated the possible effect of an endogenous ligand of benzodiazepine receptors, the triakontatetraneuropeptide [17-50] (TTN), on steroid biosynthesis in the frog hypothalamus. Immunohistochemical studies revealed that most hypothalamic neurons expressing 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase also contained peripheral-type benzodiazepine receptor-like immunoreactivity. Confocal laser scanning microscopic analysis revealed that the peripheral-type benzodiazepine receptor-immunoreactive material was located both in the cytoplasm and at the periphery of the cell bodies. By using the pulse-chase technique, TTN was found to stimulate the conversion of [3H]pregnenolone into various steroids, including 17-hydroxypregnenolone, 5 alpha-dihydrotestosterone and 17-hy...
Molecular and cellular biochemistry, 2001
Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate ... more Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Ybl, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or ...
Biochimica et biophysica acta, Jan 15, 1998
Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic d... more Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic domain that shares extensive structural similarities with the members of the sterol reductase family. To test if the sterol-reductase-like domain of LBR could be enzymatically competent, several sterol reductase-defective strains of Saccharomyces cerevisiae were transformed with a human-LBR expressing vector. LBR production did not change the ergosterol biosynthesis defect in an erg4 mutant impaired in sterol C24(28) reductase. In contrast, the sterol C14 reduction step and ergosterol prototrophy were restored in LBR-producing erg24 transformants which lack endogenous sterol C14 reductase. To test the effects of C14 reductase inhibitors on LBR activity, we constructed EMY54, an ergosterol-requiring strain that is devoid of both sterol C8-C7 isomerase and sterol C14 reductase activities. EMY54 cells recovered the capability of synthesizing ergost-8-en-3beta-ol upon transformation with a ve...
European journal of biochemistry / FEBS, Jan 15, 1989
Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular ma... more Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular mass of 34 kDa, while the minor form 2 has a molecular mass of 56 kDa. Form 1 was converted into a second major form (form 3) with a molecular mass of 68 kDa by a NAD+-dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B. (1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore sought. Both form 1 biliverdin reductases, isolated from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non-denaturing conditions and on SDS/polyacrylamide gels. ...
Journal of Biological Chemistry, 2014
The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor gr... more The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor growth and facilitates tumor invasion and metastasis. Hypoxia has been described as one trigger of angiogenesis. Indeed, hypoxia, which is characterized by areas of low oxygen levels, is a hallmark of solid tumors arising from an imbalance between oxygen delivery and consumption. Hypoxic conditions have profound effects on the different components of the tumoral environment. For example, hypoxia is able to activate endothelial cells, leading to angiogenesis but also thereby initiating a cascade of reactions involving neutrophils, smooth muscle cells, and fibroblasts. In addition, hypoxia directly regulates the expression of many genes for which the role and the importance in the tumoral environment remain to be completely elucidated. In this study, we used a method to selectively label sialoglycoproteins to identify new membrane and secreted proteins involved in the adaptative process of endothelial cells by mass spectrometry-based proteomics. We used an in vitro assay under hypoxic condition to observe an increase of protein expression or modifications of glycosylation. Then the function of the identified proteins was assessed in a vasculogenesis assay in vivo by using a morpholino strategy in zebrafish. First, our approach was validated by the identification of sialoglycoproteins such as CD105, neuropilin-1, and CLEC14A, which have already been described as playing key roles in angiogenesis. Second, we identified several new proteins regulated by hypoxia and demonstrated for the first time the pivotal role of GLUT-1, TMEM16F, and SDF4 in angiogenesis.
PLoS ONE, 2014
Glioblastoma multiform (GBM) remains clinical indication with significant ''unmet medical need''.... more Glioblastoma multiform (GBM) remains clinical indication with significant ''unmet medical need''. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.
International Journal of Peptide and Protein Research, 1976
European Journal of Biochemistry, 1998
SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative acti... more SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative activity on lymphocyte cells. Only two subtypes of sigma receptor, namely the sigma1 receptor and emopamil-binding protein, have been characterised molecularly. Only the sigma1 receptor has been shown to bind (Z)N-cyclohexyl-N-ethyl-3-(3-chloro4-cyclohexylphenyl)pro pen-2-ylamine hydrochloride (SR31747A) with high affinity. It was demonstrated that the SR31747A effect on the inhibition of T-cell proliferation was consistent with a sigma1 receptor-mediated event. In this report, binding experiments and sterol isomerase assays, using recombinant yeast strains, indicate that the recently cloned emopamil-binding protein is a new SR31747A-binding protein whose activity is inhibited by SR31747A. Sterol analyses reveal the accumulation of a delta8-cholesterol isomer at the expense of cholesterol in SR31747A-treated cells, suggesting that cholesterol biosynthesis is inhibited by SR31747A at the delta8-delta7 sterol isomerase step in animal cells. This observation is consistent with a sterol isomerase role of the emopamil-binding protein in the cholesterol biosynthetic pathway in animal cells. In contrast, there is no evidence for such a role of the sigma1 receptor, in spite of the structural similarity shared by this protein and yeast sterol isomerase. We have found that SR31747A also exerts anti-proliferative effects at nanomolar concentrations on various established cell lines. The antiproliferative activity of SR31747A is reversed by cholesterol. Sterol-isomerase overproduction enhances resistance of CHO cells. This last observation strongly suggests that sterol isomerase is implicated in the antiproliferative effect of the drug in established cell lines.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1998
Pretreatment of mouse peritoneal macrophages with interleukin-13 (IL-13) potentiates the mobiliza... more Pretreatment of mouse peritoneal macrophages with interleukin-13 (IL-13) potentiates the mobilization of arachidonic acid (AA) and the production of HETEs but does not affect the production of cyclooxygenase metabolites triggered by the suboptimal concentration of an inflammatory agonist (opsonized-zymosan). Cycloheximide suppresses these effects of IL-13 suggesting that de novo protein synthesis is involved. Indeed, IL-13 induces a time-dependent increase in the levels of cytosolic PLA2 (cPLA2) protein and mRNA. This study demonstrates a new pathway for IL-13 to modulate the inflammatory process in macrophages via modifications of cPLA2 expression and subsequent AA mobilization.
Nature, 1995
The ORL1 receptor, an orphan receptor whose human and murine complementary DNAs have recently bee... more The ORL1 receptor, an orphan receptor whose human and murine complementary DNAs have recently been characterized, structurally resembles opioid receptors and is negatively coupled with adenylate cyclase. ORL1 transcripts are particularly abundant in the central nervous system. Here we report the isolation, on the basis of its ability to inhibit the cyclase in a stable recombinant CHO(ORL1+) cell line, of a neuropeptide that resembles dynorphin A9 and whose amino acid sequence is Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln. The rat-brain cDNA encodes the peptide flanked by Lys-Arg proteolytic cleavage motifs. The synthetic heptadecapeptide potently inhibits adenylate cyclase in CHO(ORL1+) cells in culture and induces hyperalgesia when administered intracerebroventricularly to mice. Taken together, these data indicate that the newly discovered heptadecapeptide is an endogenous agonist of the ORL1 receptor and that it may be endowed with pro-nociceptive properties.
International Journal of Peptide and Protein Research, 1991
Reduced and carbamidomethylated alpaca growth hormone was submitted to tryptic digestion. Peptide... more Reduced and carbamidomethylated alpaca growth hormone was submitted to tryptic digestion. Peptides in the mixture were purified by reverse phase HPLC and N-terminal determination and an amino acid analysis of each was performed. Data obtained and the already known primary structure of the equine growth hormone allowed the assembly-by homology-of a definite sequence of amino acids for the polypeptide chain of the protein. Present data provide further information about the relationship between growth factors.
From Gene to Behavior, 2002
Yeast, 1996
In this study we used genetically manipulated strains in order to identify polypeptide spots of t... more In this study we used genetically manipulated strains in order to identify polypeptide spots of the protein map of Saccharomyces cerevisiae. Thirty-two novel polypeptide spots were identified using this strategy. They corresponded to the product of 23 different genes. We also explored the possibilities of using peptide-mass fingerprinting for the identification of proteins separated on our gels. According to this strategy, proteins contained in spots are digested with trypsin and the masses of generated peptides are determined by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). The peptide masses are then used to search a yeast protein database for proteins that match the experimental data. Application of this strategy to previously identified polypeptide spots gave evidence of the feasibility of this approach. We also report predictions on the identities of nine unknown spots using MALDI-MS.
The Plant Journal, 1998
The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD -the essent... more The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD -the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGDheptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGDheptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K d1 ≈ 1 nM, and K d2 ≈ 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.
The Journal of Cell Biology, 1999
The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular le... more The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.
European Journal of Biochemistry, 1994
Platelet factor 4 is a heparin-binding protein released from the a granules of activated platelet... more Platelet factor 4 is a heparin-binding protein released from the a granules of activated platelets. This study describes the purification and identification of two forms of rat platelet factor 4, the previously characterized non-glycosylated form of 7 kDa and an additional glycosylated form of molecular mass 9 kDa. The two proteins both neutralized the antithrombin-111-dependent inhibitory activity of heparin. Although their amino acid composition was found to be the same, in the Nterminal sequence of the 9-kDa protein, the second threonine residue could not be detected and a difference of 976Da was determined by mass spectrometry. After digestion with 0-glycanase and sialidase, the two proteins showed the same molecular mass. Overall consideration of these data led to identification of the higher-molecular-mass protein as a glycosylated form of rat platelet factor 4 with 0-glycosylation at the second N-terminal amino acid, while the structure of the oligosaccharide core was established by mass spectrometry and sugar differentiation with lectins. The two forms of platelet factor 4 are both present in platelets and secreted after platelet activation.
The FASEB Journal, 2014
Dysfunctions in Wnt signaling increase β-catenin stability and are associated with cancers, inclu... more Dysfunctions in Wnt signaling increase β-catenin stability and are associated with cancers, including colorectal cancer. In addition, β-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of β-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of β-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of β-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of β-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for β-catenin stability. Analyses of β-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the β-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the β-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of β-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of β-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.
Proceedings of the National Academy of Sciences, 2006
P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been rec... more P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12͞P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met. mechanism of action ͉ platelet ͉ antiaggregant Conflict of interest statement:
Preparative Biochemistry, 1989
Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular d... more Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developed. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.
Oncogene, 2006
Natural killer cells are well known to play an important role in immune defense against tumor dev... more Natural killer cells are well known to play an important role in immune defense against tumor development and viral infections. To further characterize new functionally relevant structures in these cells, we studied a series of monoclonal antibodies that we have raised against the NK cell line YT. One of these antibodies previously described as AY19, recognizes a 85 kD surface glycoprotein. Here we report the identification of a new secreted isoform of protocadherin 15, PCDH15C, which represents a potential associated protein for p85. Importantly, whereas protocadherins are absent from the surface of normal hematopoietic cells, we describe, for the first time, that PCDH15 is expressed in cytotoxic tumor-derived T-and NK-cell lines as well as in biopsies of nasal NK/T-cell lymphomas.
Neuroscience, 1998
Neurons and glial cells are capable of synthesizing various bioactive steroids, but the neuronal ... more Neurons and glial cells are capable of synthesizing various bioactive steroids, but the neuronal mechanisms controlling neurosteroid-secreting cells are poorly understood. In the present study, we have investigated the possible effect of an endogenous ligand of benzodiazepine receptors, the triakontatetraneuropeptide [17-50] (TTN), on steroid biosynthesis in the frog hypothalamus. Immunohistochemical studies revealed that most hypothalamic neurons expressing 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase also contained peripheral-type benzodiazepine receptor-like immunoreactivity. Confocal laser scanning microscopic analysis revealed that the peripheral-type benzodiazepine receptor-immunoreactive material was located both in the cytoplasm and at the periphery of the cell bodies. By using the pulse-chase technique, TTN was found to stimulate the conversion of [3H]pregnenolone into various steroids, including 17-hydroxypregnenolone, 5 alpha-dihydrotestosterone and 17-hy...
Molecular and cellular biochemistry, 2001
Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate ... more Two-dimensional non-equilibrium pH gel electrophoresis (2D-NEPHGE) analysis was used to evaluate the effects of dietary protein depletion on the protein composition of mouse liver cytosol. Analysing the cytosol from both normal and protein depleted liver, the position in gels of more than three hundred protein spots was determined. After 5 days of protein depletion, about 20% of the spots either increased or decreased more than 2 fold. Five spots of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recognised by specific antibodies. The glutathione S-transferase (GSTs) subunits Ybl, Yc and Yf were identified by the simultaneous analysis of both glutathione-binding cytosolic proteins and the corresponding standards. As estimated by internal optical density (IOD) of spots, the changes caused by protein depletion in GAPDH and GST subunit contents were similar to those obtained by other methods. By means of mass spectrometric analysis of tryptic peptides generated from spots and/or ...
Biochimica et biophysica acta, Jan 15, 1998
Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic d... more Lamin B receptor (LBR), a nuclear protein of avian and mammalian cells, contains an hydrophobic domain that shares extensive structural similarities with the members of the sterol reductase family. To test if the sterol-reductase-like domain of LBR could be enzymatically competent, several sterol reductase-defective strains of Saccharomyces cerevisiae were transformed with a human-LBR expressing vector. LBR production did not change the ergosterol biosynthesis defect in an erg4 mutant impaired in sterol C24(28) reductase. In contrast, the sterol C14 reduction step and ergosterol prototrophy were restored in LBR-producing erg24 transformants which lack endogenous sterol C14 reductase. To test the effects of C14 reductase inhibitors on LBR activity, we constructed EMY54, an ergosterol-requiring strain that is devoid of both sterol C8-C7 isomerase and sterol C14 reductase activities. EMY54 cells recovered the capability of synthesizing ergost-8-en-3beta-ol upon transformation with a ve...
European journal of biochemistry / FEBS, Jan 15, 1989
Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular ma... more Rat-liver biliverdin reductase exists in two molecular forms. The major form 1 has a molecular mass of 34 kDa, while the minor form 2 has a molecular mass of 56 kDa. Form 1 was converted into a second major form (form 3) with a molecular mass of 68 kDa by a NAD+-dependent peroxisomal dehydrogenase which was induced under conditions of oxidative stress [Frydman, R. B., Tomaro, M. L., Awruch, J. & Frydman, B. (1984) Biochem. Biophys. Res. Commun. 121, 249]. Molecular form 1 from rat kidney was not affected by the dehydrogenase, and a structural explanation for this difference was therefore sought. Both form 1 biliverdin reductases, isolated from rat liver and kidney, were purified to homogeneity using affinity chromatography, FPLC and HPLC techniques. The homogeneous enzymes were found to be identical when compared by their HPLC retention times, amino acid compositions and electrophoretic behaviour on polyacrylamide gels under non-denaturing conditions and on SDS/polyacrylamide gels. ...
Journal of Biological Chemistry, 2014
The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor gr... more The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor growth and facilitates tumor invasion and metastasis. Hypoxia has been described as one trigger of angiogenesis. Indeed, hypoxia, which is characterized by areas of low oxygen levels, is a hallmark of solid tumors arising from an imbalance between oxygen delivery and consumption. Hypoxic conditions have profound effects on the different components of the tumoral environment. For example, hypoxia is able to activate endothelial cells, leading to angiogenesis but also thereby initiating a cascade of reactions involving neutrophils, smooth muscle cells, and fibroblasts. In addition, hypoxia directly regulates the expression of many genes for which the role and the importance in the tumoral environment remain to be completely elucidated. In this study, we used a method to selectively label sialoglycoproteins to identify new membrane and secreted proteins involved in the adaptative process of endothelial cells by mass spectrometry-based proteomics. We used an in vitro assay under hypoxic condition to observe an increase of protein expression or modifications of glycosylation. Then the function of the identified proteins was assessed in a vasculogenesis assay in vivo by using a morpholino strategy in zebrafish. First, our approach was validated by the identification of sialoglycoproteins such as CD105, neuropilin-1, and CLEC14A, which have already been described as playing key roles in angiogenesis. Second, we identified several new proteins regulated by hypoxia and demonstrated for the first time the pivotal role of GLUT-1, TMEM16F, and SDF4 in angiogenesis.
PLoS ONE, 2014
Glioblastoma multiform (GBM) remains clinical indication with significant ''unmet medical need''.... more Glioblastoma multiform (GBM) remains clinical indication with significant ''unmet medical need''. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.
International Journal of Peptide and Protein Research, 1976
European Journal of Biochemistry, 1998
SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative acti... more SR31747A is a new sigma ligand exhibiting immunosuppressive properties and antiproliferative activity on lymphocyte cells. Only two subtypes of sigma receptor, namely the sigma1 receptor and emopamil-binding protein, have been characterised molecularly. Only the sigma1 receptor has been shown to bind (Z)N-cyclohexyl-N-ethyl-3-(3-chloro4-cyclohexylphenyl)pro pen-2-ylamine hydrochloride (SR31747A) with high affinity. It was demonstrated that the SR31747A effect on the inhibition of T-cell proliferation was consistent with a sigma1 receptor-mediated event. In this report, binding experiments and sterol isomerase assays, using recombinant yeast strains, indicate that the recently cloned emopamil-binding protein is a new SR31747A-binding protein whose activity is inhibited by SR31747A. Sterol analyses reveal the accumulation of a delta8-cholesterol isomer at the expense of cholesterol in SR31747A-treated cells, suggesting that cholesterol biosynthesis is inhibited by SR31747A at the delta8-delta7 sterol isomerase step in animal cells. This observation is consistent with a sterol isomerase role of the emopamil-binding protein in the cholesterol biosynthetic pathway in animal cells. In contrast, there is no evidence for such a role of the sigma1 receptor, in spite of the structural similarity shared by this protein and yeast sterol isomerase. We have found that SR31747A also exerts anti-proliferative effects at nanomolar concentrations on various established cell lines. The antiproliferative activity of SR31747A is reversed by cholesterol. Sterol-isomerase overproduction enhances resistance of CHO cells. This last observation strongly suggests that sterol isomerase is implicated in the antiproliferative effect of the drug in established cell lines.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1998
Pretreatment of mouse peritoneal macrophages with interleukin-13 (IL-13) potentiates the mobiliza... more Pretreatment of mouse peritoneal macrophages with interleukin-13 (IL-13) potentiates the mobilization of arachidonic acid (AA) and the production of HETEs but does not affect the production of cyclooxygenase metabolites triggered by the suboptimal concentration of an inflammatory agonist (opsonized-zymosan). Cycloheximide suppresses these effects of IL-13 suggesting that de novo protein synthesis is involved. Indeed, IL-13 induces a time-dependent increase in the levels of cytosolic PLA2 (cPLA2) protein and mRNA. This study demonstrates a new pathway for IL-13 to modulate the inflammatory process in macrophages via modifications of cPLA2 expression and subsequent AA mobilization.
Nature, 1995
The ORL1 receptor, an orphan receptor whose human and murine complementary DNAs have recently bee... more The ORL1 receptor, an orphan receptor whose human and murine complementary DNAs have recently been characterized, structurally resembles opioid receptors and is negatively coupled with adenylate cyclase. ORL1 transcripts are particularly abundant in the central nervous system. Here we report the isolation, on the basis of its ability to inhibit the cyclase in a stable recombinant CHO(ORL1+) cell line, of a neuropeptide that resembles dynorphin A9 and whose amino acid sequence is Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln. The rat-brain cDNA encodes the peptide flanked by Lys-Arg proteolytic cleavage motifs. The synthetic heptadecapeptide potently inhibits adenylate cyclase in CHO(ORL1+) cells in culture and induces hyperalgesia when administered intracerebroventricularly to mice. Taken together, these data indicate that the newly discovered heptadecapeptide is an endogenous agonist of the ORL1 receptor and that it may be endowed with pro-nociceptive properties.
International Journal of Peptide and Protein Research, 1991
Reduced and carbamidomethylated alpaca growth hormone was submitted to tryptic digestion. Peptide... more Reduced and carbamidomethylated alpaca growth hormone was submitted to tryptic digestion. Peptides in the mixture were purified by reverse phase HPLC and N-terminal determination and an amino acid analysis of each was performed. Data obtained and the already known primary structure of the equine growth hormone allowed the assembly-by homology-of a definite sequence of amino acids for the polypeptide chain of the protein. Present data provide further information about the relationship between growth factors.
From Gene to Behavior, 2002
Yeast, 1996
In this study we used genetically manipulated strains in order to identify polypeptide spots of t... more In this study we used genetically manipulated strains in order to identify polypeptide spots of the protein map of Saccharomyces cerevisiae. Thirty-two novel polypeptide spots were identified using this strategy. They corresponded to the product of 23 different genes. We also explored the possibilities of using peptide-mass fingerprinting for the identification of proteins separated on our gels. According to this strategy, proteins contained in spots are digested with trypsin and the masses of generated peptides are determined by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). The peptide masses are then used to search a yeast protein database for proteins that match the experimental data. Application of this strategy to previously identified polypeptide spots gave evidence of the feasibility of this approach. We also report predictions on the identities of nine unknown spots using MALDI-MS.
The Plant Journal, 1998
The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD -the essent... more The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD -the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGDheptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGDheptapeptide. The binding is saturable, reversible, and two types of high affinity sites (K d1 ≈ 1 nM, and K d2 ≈ 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.
The Journal of Cell Biology, 1999
The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular le... more The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.
European Journal of Biochemistry, 1994
Platelet factor 4 is a heparin-binding protein released from the a granules of activated platelet... more Platelet factor 4 is a heparin-binding protein released from the a granules of activated platelets. This study describes the purification and identification of two forms of rat platelet factor 4, the previously characterized non-glycosylated form of 7 kDa and an additional glycosylated form of molecular mass 9 kDa. The two proteins both neutralized the antithrombin-111-dependent inhibitory activity of heparin. Although their amino acid composition was found to be the same, in the Nterminal sequence of the 9-kDa protein, the second threonine residue could not be detected and a difference of 976Da was determined by mass spectrometry. After digestion with 0-glycanase and sialidase, the two proteins showed the same molecular mass. Overall consideration of these data led to identification of the higher-molecular-mass protein as a glycosylated form of rat platelet factor 4 with 0-glycosylation at the second N-terminal amino acid, while the structure of the oligosaccharide core was established by mass spectrometry and sugar differentiation with lectins. The two forms of platelet factor 4 are both present in platelets and secreted after platelet activation.
The FASEB Journal, 2014
Dysfunctions in Wnt signaling increase β-catenin stability and are associated with cancers, inclu... more Dysfunctions in Wnt signaling increase β-catenin stability and are associated with cancers, including colorectal cancer. In addition, β-catenin degradation is decreased by nutrient-dependent O-GlcNAcylation. Human colon tumors and colons from mice fed high-carbohydrate diets exhibited higher amounts of β-catenin and O-GlcNAc relative to healthy tissues and mice fed a standard diet, respectively. Administration of the O-GlcNAcase inhibitor thiamet G to mice also increased colonic expression of β-catenin. By ETD-MS/MS, we identified 4 O-GlcNAcylation sites at the N terminus of β-catenin (S23/T40/T41/T112). Furthermore, mutation of serine and threonine residues within the D box of β-catenin reduced O-GlcNAcylation by 75%. Interestingly, elevating O-GlcNAcylation in human colon cell lines drastically reduced phosphorylation at T41, a key residue of the D box responsible for β-catenin stability. Analyses of β-catenin O-GlcNAcylation mutants reinforced T41 as the most crucial residue that controls the β-catenin degradation rate. Finally, inhibiting O-GlcNAcylation decreased the β-catenin/α-catenin interaction necessary for mucosa integrity, whereas O-GlcNAcase silencing improved this interaction. These results suggest that O-GlcNAcylation regulates not only the stability of β-catenin, but also affects its localization at the level of adherens junctions. Accordingly, we propose that O-GlcNAcylation of β-catenin is a missing link between the glucose metabolism deregulation observed in metabolic disorders and the development of cancer.
Proceedings of the National Academy of Sciences, 2006
P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been rec... more P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12͞P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met. mechanism of action ͉ platelet ͉ antiaggregant Conflict of interest statement:
Preparative Biochemistry, 1989
Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular d... more Rat cystatin C was purified to apparent homogeneity from rat urine after induction of a tubular dysfunction with sodium chromate. Twentyfold concentrated urine was chromatographed by a rapid purification procedure. A two-step purification including affinity chromatography on carboxymethyl papain- Sepharose and high-resolution anion exchange chromatography was developed. The purified protein has an apparent molecular mass of 15 kDa and pI of 10.2; its aminoacid composition was similar to human cystatin C. As opposed to previous data, purified urinary rat cystatin C did not contain significant amounts of carbohydrate. Antisera against rat cystatin C, raised in rabbits, partially cross-reacted with human and mouse cystatin C, indicating their antigenic similarities. Like human cystatin C, native rat cystatin C, named slow form, is degraded into a more acidic form, called fast form, by a loss of N-terminal amino acids; fast form displayed a pI of 9.4.
Oncogene, 2006
Natural killer cells are well known to play an important role in immune defense against tumor dev... more Natural killer cells are well known to play an important role in immune defense against tumor development and viral infections. To further characterize new functionally relevant structures in these cells, we studied a series of monoclonal antibodies that we have raised against the NK cell line YT. One of these antibodies previously described as AY19, recognizes a 85 kD surface glycoprotein. Here we report the identification of a new secreted isoform of protocadherin 15, PCDH15C, which represents a potential associated protein for p85. Importantly, whereas protocadherins are absent from the surface of normal hematopoietic cells, we describe, for the first time, that PCDH15 is expressed in cytotoxic tumor-derived T-and NK-cell lines as well as in biopsies of nasal NK/T-cell lymphomas.