Saïd Taouji - Academia.edu (original) (raw)

Papers by Saïd Taouji

Research paper thumbnail of AlphaScreen®-Based Characterization of the Bifunctional Kinase/RNase IRE1α: A Novel and Atypical Drug Target

assay technologies that were originally developed for high-throughput screening (hts) have recent... more assay technologies that were originally developed for high-throughput screening (hts) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (adMet) of hts. here the authors investigated and characterized the biological properties of a novel target, ire1α, a bifunctional kinase/rnase stress sensor of the endoplasmic reticulum (er). they have developed a novel assay platform using the hts technology alphascreen ® to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of ire1α. they show in vitro that dimerization/oligomerization of the cytosolic domain of ire1 correlated with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mrna. using orthogonal in vitro and cell-based approaches, the authors show that the results obtained using alphascreen ® were biologically relevant. Preliminary characterization of assay robustness indicates that both alphascreen ® assays should be useful in hts for the identification of ire1 activity modulators.

Research paper thumbnail of Epidemiology and prophylaxis of neonatal diarrhoea in the foal - preliminary results of a study carried out in Normandy

Research paper thumbnail of Supplementary Figure 2 from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Research paper thumbnail of Supplementary Table 1 from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Research paper thumbnail of Supplementary Methods from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Research paper thumbnail of Supplementary Tables 1 - 5, Figures 1 - 11 from Posttranscriptional Regulation of <i>PER1</i> Underlies the Oncogenic Function of IREα

Research paper thumbnail of Supplementary Table 2 from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

PDF file - 36K, Quantification of eiF2α phosphorylation upon sorafenib treatment. X-ray film were... more PDF file - 36K, Quantification of eiF2α phosphorylation upon sorafenib treatment. X-ray film were quantified using scanning densitometry and data are represented as arbitrary units � SD.

Research paper thumbnail of Supplementary Figure 1 from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Research paper thumbnail of Supplementary Figure Legend from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Supplementary Table 2-Quantification of eiF2α phosphorylation upon sorafenib treatment. X-ray fil... more Supplementary Table 2-Quantification of eiF2α phosphorylation upon sorafenib treatment. X-ray film were quantified using scanning densitometry and data are represented as arbitrary units ± SD. Figure S1. (A) Quantification of Xbp1 mRNA splicing shown in Figure 2A. (B) Wild-type HepG2 cells were either not treated or treated with 10 µM Sorafenib for 2 h followed by staining for giantin, a Golgi complex marker, α-tubulin, as a marker for the microtubules and Hoechst 33342 as a nuclear marker. (C) Cells treated as above were immunostained against giantin and phalloidin was used to stain the actin cytoskeleton. Images were acquired by wide-field fluorescence microscopy. Scale bars correspond to 25 µm.

Research paper thumbnail of Genome‐wide screen identifies a novel p97/ <scp>CDC</scp> ‐48‐dependent pathway regulating <scp>ER</scp> ‐stress‐induced gene transcription

EMBO Reports, Feb 4, 2015

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded P... more The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR ER) to restore ER homeostasis. The AAA + ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR ER genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA + ATPase, as a novel repressor of a subset of UPR ER genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR ER genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.

Research paper thumbnail of Posttranscriptional Regulation of <i>PER1</i> Underlies the Oncogenic Function of IREα

Cancer Research, Jul 31, 2013

Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; howev... more Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1a. Analysis of the mechanism shows that IRE1a endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1a signaling using either siRNA-mediated silencing or a dominantnegative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-a substrate, thereby pointing toward an increased IRE1a activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development. Cancer Res; 73(15); 4732-43. Ó2013 AACR.

Research paper thumbnail of Immunization of mares against the antigens of Rhodococcus equi: evaluation of the immunity response and passive transfer in differents stables in Normandy

Research paper thumbnail of 2001. Characterization of mutations in the rpoB gene associated with rifampin resistance in Rhodococcus equi isolated from foals

Treatment with a combination of erythromycin and rifampin has considerably improved survival rate... more Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by Rhodococcus equi. Fre-quently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rifr)-determining regions of 12 rifampin-resistant R. equi strains isolated from three foals and of mutants selected in vitro from R. equi ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible R. equi strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (Escherichia coli numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 mg/ml), whereas His526Asp conferred high-level resistance (rifam-pin MIC, 128 mg/ml) in the 7 remain...

Research paper thumbnail of Data from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the ... more The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer-derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal-regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA þ ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients. Mol Cancer Ther; 11(12); 2610-20. Ó2012 AACR.

Research paper thumbnail of 2012 Yi MCT

Research paper thumbnail of Foal IgG and opsonizing anti- antibodies after immunization of pregnant mares with a protective VapA candidate vaccine

Research paper thumbnail of Vaccine Composition Against Rhodococcus Equi

Research paper thumbnail of Structure et fonction des glycoproteines de la cellule de kurloff. Relation avec l'activite natural killer au cours d'une leucemie experimentale

Http Www Theses Fr, 1996

Les proprietes anti-tumorales de la cellule de kurloff, cellule oestrogenodependante a activite n... more Les proprietes anti-tumorales de la cellule de kurloff, cellule oestrogenodependante a activite natural killer, ont ete etudiees dans un modele de leucemie experimentale chez le cobaye s2. L'injection, par voie intra-cardiaque, de cellules leucemiques l2c, provoque une augmentation significative du nombre de cellules de kurloff dans le sang des cobayes s2 oestrogenises, les cellules de kurloff semblent avoir un role dans le controle du developpement de la leucemie puisque le nombre d'animaux leucemiques chute de 50% lorsqu'ils sont oestregenises. Durant la phase initiale du developpement de la leucemie, nous avons egalement observe: 1) que la distribution tissulaire des cellules de kurloff etant modifiee, 2) que parallelement a une augmentation de l'activite arylsulfatasique, seules les isoformes acides etaient exprimees, et enfain 3 un accroissement significatif des activites phosphatasiques acides et proteasiques ainsi que du taux d'acide sialique dans les glycoconjugues. Les arylsulfatases de la cellule de kurloff, enzymes lysosomiques du type iib, se caracterisent par la presence de deux populations de 55 et 62 kda, se distinguant par leur point isoelectrique acide (pi 4,4-4,6) et basique (pi 8,5). La forte anionicite des isoformes acides est due a des groupements phosphates. Les phosphatases acides de la cellule de kurloff sont n-glycosylproteines de 200 et 500 kda, existant sous la forme de plusieurs isoenzymes. Apres leur purification, les oligosaccharides (11% en masse) des sialphosphatases acides (heterodimeres de 33 kda et 36 kda, sensible au tartrate) ont ete analyses par chromatographie d'affinite, par lectine-blotting ainsi que par des glycosidases. Par lectine-histochimie et par western-blotting ainsi que par l'utilisation de sialidases specifiques, l'existence de o-glycosylproteines possedant des sequences lactosaminiques sialysees en alpha-2,6 mais egalement polysialylees en alpha-2,8 a ete demontree en meme temps que la presence de n-polysialloproteines

Research paper thumbnail of Serum and mucosal antibodies of infected foals recognized two distinct epitopes of VapA ofRhodococcus equi

FEMS Immunology & Medical Microbiology, 2002

Virulence-associated protein A (VapA) of Rhodococcus equi has been proposed for use both as a vac... more Virulence-associated protein A (VapA) of Rhodococcus equi has been proposed for use both as a vaccine and as a target for antibodies in immunotherapy and diagnostic tests. Epitope mapping of VapA allowed the identification of two B cell epitopes associated with R. equi pneumonia. The peptide NLQKDEPGRASDT was confirmed as an immunodominant N-terminal B cell epitope recognized by all sera from infected foals while VSFQYNAVGPYLNINFFDSS (C-terminal B cell epitope) was exclusively recognized by IgA from the tracheal aspirates. Moreover, specific antibodies produced against the VapA-specific peptide reacted with a major protein (approximately 20 kDa) from R. equi antigens separated by two-dimensional gel electrophoresis. The strong reactivity of mucosal IgA from infected foals with the conserved peptides might constitute an attractive target for diagnosis and vaccine.

Research paper thumbnail of First identification of small-molecule inhibitors of Pontin by combining virtual screening and enzymatic assay

Biochemical Journal, 2012

The human protein Pontin, which belongs to the AAA+ (ATPases associated with various cellular act... more The human protein Pontin, which belongs to the AAA+ (ATPases associated with various cellular activities) family, is overexpressed in several cancers and its silencing in vitro leads to tumour cell growth arrest and apoptosis, making it a good target for cancer therapy. In particular, high levels of expression were found in hepatic tumours for which the therapeutic arsenal is rather limited. The three-dimensional structure of Pontin has been resolved previously, revealing a hexameric assembly with one ADP molecule co-crystallized in each subunit. Using Vina, DrugScore and Xscore, structure-based virtual screening of 2200 commercial molecules was conducted into the ATP-binding site formed by a dimer of Pontin in order to prioritize the best candidates. Complementary to the in silico screening, a versatile and sensitive colorimetric assay was set up to measure the disruption of the ATPase activity of Pontin. This assay allowed the determination of inhibition curves for more than 20 to...

Research paper thumbnail of AlphaScreen®-Based Characterization of the Bifunctional Kinase/RNase IRE1α: A Novel and Atypical Drug Target

assay technologies that were originally developed for high-throughput screening (hts) have recent... more assay technologies that were originally developed for high-throughput screening (hts) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (adMet) of hts. here the authors investigated and characterized the biological properties of a novel target, ire1α, a bifunctional kinase/rnase stress sensor of the endoplasmic reticulum (er). they have developed a novel assay platform using the hts technology alphascreen ® to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of ire1α. they show in vitro that dimerization/oligomerization of the cytosolic domain of ire1 correlated with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mrna. using orthogonal in vitro and cell-based approaches, the authors show that the results obtained using alphascreen ® were biologically relevant. Preliminary characterization of assay robustness indicates that both alphascreen ® assays should be useful in hts for the identification of ire1 activity modulators.

Research paper thumbnail of Epidemiology and prophylaxis of neonatal diarrhoea in the foal - preliminary results of a study carried out in Normandy

Research paper thumbnail of Supplementary Figure 2 from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Research paper thumbnail of Supplementary Table 1 from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Research paper thumbnail of Supplementary Methods from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Research paper thumbnail of Supplementary Tables 1 - 5, Figures 1 - 11 from Posttranscriptional Regulation of <i>PER1</i> Underlies the Oncogenic Function of IREα

Research paper thumbnail of Supplementary Table 2 from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

PDF file - 36K, Quantification of eiF2α phosphorylation upon sorafenib treatment. X-ray film were... more PDF file - 36K, Quantification of eiF2α phosphorylation upon sorafenib treatment. X-ray film were quantified using scanning densitometry and data are represented as arbitrary units � SD.

Research paper thumbnail of Supplementary Figure 1 from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Research paper thumbnail of Supplementary Figure Legend from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

Supplementary Table 2-Quantification of eiF2α phosphorylation upon sorafenib treatment. X-ray fil... more Supplementary Table 2-Quantification of eiF2α phosphorylation upon sorafenib treatment. X-ray film were quantified using scanning densitometry and data are represented as arbitrary units ± SD. Figure S1. (A) Quantification of Xbp1 mRNA splicing shown in Figure 2A. (B) Wild-type HepG2 cells were either not treated or treated with 10 µM Sorafenib for 2 h followed by staining for giantin, a Golgi complex marker, α-tubulin, as a marker for the microtubules and Hoechst 33342 as a nuclear marker. (C) Cells treated as above were immunostained against giantin and phalloidin was used to stain the actin cytoskeleton. Images were acquired by wide-field fluorescence microscopy. Scale bars correspond to 25 µm.

Research paper thumbnail of Genome‐wide screen identifies a novel p97/ <scp>CDC</scp> ‐48‐dependent pathway regulating <scp>ER</scp> ‐stress‐induced gene transcription

EMBO Reports, Feb 4, 2015

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded P... more The accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates the Unfolded Protein Response (UPR ER) to restore ER homeostasis. The AAA + ATPase p97/CDC-48 plays key roles in ER stress by promoting both ER protein degradation and transcription of UPR ER genes. Although the mechanisms associated with protein degradation are now well established, the molecular events involved in the regulation of gene transcription by p97/CDC-48 remain unclear. Using a reporter-based genome-wide RNAi screen in combination with quantitative proteomic analysis in Caenorhabditis elegans, we have identified RUVB-2, a AAA + ATPase, as a novel repressor of a subset of UPR ER genes. We show that degradation of RUVB-2 by CDC-48 enhances expression of ER stress response genes through an XBP1-dependent mechanism. The functional interplay between CDC-48 and RUVB-2 in controlling transcription of select UPR ER genes appears conserved in human cells. Together, these results describe a novel role for p97/CDC-48, whereby its role in protein degradation is integrated with its role in regulating expression of ER stress response genes.

Research paper thumbnail of Posttranscriptional Regulation of <i>PER1</i> Underlies the Oncogenic Function of IREα

Cancer Research, Jul 31, 2013

Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; howev... more Growing evidence supports a role for the unfolded protein response (UPR) in carcinogenesis; however, the precise molecular mechanisms underlying this phenomenon remain elusive. Herein, we identified the circadian clock PER1 mRNA as a novel substrate of the endoribonuclease activity of the UPR sensor IRE1a. Analysis of the mechanism shows that IRE1a endoribonuclease activity decreased PER1 mRNA in tumor cells without affecting PER1 gene transcription. Inhibition of IRE1a signaling using either siRNA-mediated silencing or a dominantnegative strategy prevented PER1 mRNA decay, reduced tumorigenesis, and increased survival, features that were reversed upon PER1 silencing. Clinically, patients showing reduced survival have lower levels of PER1 mRNA expression and increased splicing of XBP1, a known IRE-a substrate, thereby pointing toward an increased IRE1a activity in these patients. Hence, we describe a novel mechanism connecting the UPR and circadian clock components in tumor cells, thereby highlighting the importance of this interplay in tumor development. Cancer Res; 73(15); 4732-43. Ó2013 AACR.

Research paper thumbnail of Immunization of mares against the antigens of Rhodococcus equi: evaluation of the immunity response and passive transfer in differents stables in Normandy

Research paper thumbnail of 2001. Characterization of mutations in the rpoB gene associated with rifampin resistance in Rhodococcus equi isolated from foals

Treatment with a combination of erythromycin and rifampin has considerably improved survival rate... more Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by Rhodococcus equi. Fre-quently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rifr)-determining regions of 12 rifampin-resistant R. equi strains isolated from three foals and of mutants selected in vitro from R. equi ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible R. equi strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (Escherichia coli numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 mg/ml), whereas His526Asp conferred high-level resistance (rifam-pin MIC, 128 mg/ml) in the 7 remain...

Research paper thumbnail of Data from Sorafenib-Mediated Targeting of the AAA<sup>+</sup> ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the ... more The molecular mechanisms and cellular targets of sorafenib, a multikinase inhibitor used for the treatment of hepatocellular carcinoma (HCC), remain to be fully characterized. Recent studies have shown that sorafenib induces tumor cell death through the activation of endoplasmic reticulum stress signaling and/or autophagy in various cellular models. Using liver cancer-derived cell lines, we specifically show that the IRE1 and phosphorylated extracellular signal-regulated kinase arms of the unfolded protein response (UPR) become activated upon sorafenib treatment, whereas the ATF6 arm is inhibited. Our results also reveal that sorafenib treatment causes disruption to the secretory pathway, as witnessed by the fragmentation of the Golgi apparatus and the induction of autophagy. On the basis of these observations, we tested the relevance of the AAA þ ATPase p97/VCP as a potential functional target of sorafenib. Our results show that p97/VCP tyrosine phosphorylation is prevented upon sorafenib treatment, and that this can be correlated with enhanced membrane association. Moreover, we show that DBeQ, a recently discovered inhibitor of p97/VCP, enhances sorafenib-mediated toxicity in cultured cells. Our data show a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we identify p97/VCP phosphorylation as a potential target for improved sorafenib treatment efficacy in patients. Mol Cancer Ther; 11(12); 2610-20. Ó2012 AACR.

Research paper thumbnail of 2012 Yi MCT

Research paper thumbnail of Foal IgG and opsonizing anti- antibodies after immunization of pregnant mares with a protective VapA candidate vaccine

Research paper thumbnail of Vaccine Composition Against Rhodococcus Equi

Research paper thumbnail of Structure et fonction des glycoproteines de la cellule de kurloff. Relation avec l'activite natural killer au cours d'une leucemie experimentale

Http Www Theses Fr, 1996

Les proprietes anti-tumorales de la cellule de kurloff, cellule oestrogenodependante a activite n... more Les proprietes anti-tumorales de la cellule de kurloff, cellule oestrogenodependante a activite natural killer, ont ete etudiees dans un modele de leucemie experimentale chez le cobaye s2. L'injection, par voie intra-cardiaque, de cellules leucemiques l2c, provoque une augmentation significative du nombre de cellules de kurloff dans le sang des cobayes s2 oestrogenises, les cellules de kurloff semblent avoir un role dans le controle du developpement de la leucemie puisque le nombre d'animaux leucemiques chute de 50% lorsqu'ils sont oestregenises. Durant la phase initiale du developpement de la leucemie, nous avons egalement observe: 1) que la distribution tissulaire des cellules de kurloff etant modifiee, 2) que parallelement a une augmentation de l'activite arylsulfatasique, seules les isoformes acides etaient exprimees, et enfain 3 un accroissement significatif des activites phosphatasiques acides et proteasiques ainsi que du taux d'acide sialique dans les glycoconjugues. Les arylsulfatases de la cellule de kurloff, enzymes lysosomiques du type iib, se caracterisent par la presence de deux populations de 55 et 62 kda, se distinguant par leur point isoelectrique acide (pi 4,4-4,6) et basique (pi 8,5). La forte anionicite des isoformes acides est due a des groupements phosphates. Les phosphatases acides de la cellule de kurloff sont n-glycosylproteines de 200 et 500 kda, existant sous la forme de plusieurs isoenzymes. Apres leur purification, les oligosaccharides (11% en masse) des sialphosphatases acides (heterodimeres de 33 kda et 36 kda, sensible au tartrate) ont ete analyses par chromatographie d'affinite, par lectine-blotting ainsi que par des glycosidases. Par lectine-histochimie et par western-blotting ainsi que par l'utilisation de sialidases specifiques, l'existence de o-glycosylproteines possedant des sequences lactosaminiques sialysees en alpha-2,6 mais egalement polysialylees en alpha-2,8 a ete demontree en meme temps que la presence de n-polysialloproteines

Research paper thumbnail of Serum and mucosal antibodies of infected foals recognized two distinct epitopes of VapA ofRhodococcus equi

FEMS Immunology & Medical Microbiology, 2002

Virulence-associated protein A (VapA) of Rhodococcus equi has been proposed for use both as a vac... more Virulence-associated protein A (VapA) of Rhodococcus equi has been proposed for use both as a vaccine and as a target for antibodies in immunotherapy and diagnostic tests. Epitope mapping of VapA allowed the identification of two B cell epitopes associated with R. equi pneumonia. The peptide NLQKDEPGRASDT was confirmed as an immunodominant N-terminal B cell epitope recognized by all sera from infected foals while VSFQYNAVGPYLNINFFDSS (C-terminal B cell epitope) was exclusively recognized by IgA from the tracheal aspirates. Moreover, specific antibodies produced against the VapA-specific peptide reacted with a major protein (approximately 20 kDa) from R. equi antigens separated by two-dimensional gel electrophoresis. The strong reactivity of mucosal IgA from infected foals with the conserved peptides might constitute an attractive target for diagnosis and vaccine.

Research paper thumbnail of First identification of small-molecule inhibitors of Pontin by combining virtual screening and enzymatic assay

Biochemical Journal, 2012

The human protein Pontin, which belongs to the AAA+ (ATPases associated with various cellular act... more The human protein Pontin, which belongs to the AAA+ (ATPases associated with various cellular activities) family, is overexpressed in several cancers and its silencing in vitro leads to tumour cell growth arrest and apoptosis, making it a good target for cancer therapy. In particular, high levels of expression were found in hepatic tumours for which the therapeutic arsenal is rather limited. The three-dimensional structure of Pontin has been resolved previously, revealing a hexameric assembly with one ADP molecule co-crystallized in each subunit. Using Vina, DrugScore and Xscore, structure-based virtual screening of 2200 commercial molecules was conducted into the ATP-binding site formed by a dimer of Pontin in order to prioritize the best candidates. Complementary to the in silico screening, a versatile and sensitive colorimetric assay was set up to measure the disruption of the ATPase activity of Pontin. This assay allowed the determination of inhibition curves for more than 20 to...