Xavier Espanel - Academia.edu (original) (raw)
Papers by Xavier Espanel
HAL (Le Centre pour la Communication Scientifique Directe), Oct 1, 1998
Retinoblastoma (Rb) protein has been implicated in the control of cell proliferation and malignan... more Retinoblastoma (Rb) protein has been implicated in the control of cell proliferation and malignant transformation in different cell types. To analyze ifs role as a promoter of cell growth arrest during development, we have studied the temporal pattern of Rb expression and its association with E2F-1 during embryogenesis of the quail neuroretina. During development of this neural organ, moat cells stop dividing and begin to differentiate at embryonic days E6 and E7, as indicated by the decline of cyclindependent kinase cdk2 and by an increased level of cdk5. At this stage, we observed a shift of hyperphosphorylated Rb protein to Its hypophosphorylated form as well as a decrease of the total level of Rb. The Rb-related protein, p107, is also progressively down-regulated from the E7 stage onwards. P130 levels, on the other hand, actually increase. Moreover, cell cycle exit at E6-E7 is characterized by a sudden and transient rise of the E2F-1/RB complex followed by the appearance of the E2F-4/p130 complex starting at E8. Conversely, expression of adenovirus EIA protein in E6 neuroretina cells leads to a dissociation of E2F-1/Rb complex and suppression of cell growth arrest and differentiation. This suggests that cell cycle exit and re-entry may depend on Rb/E2F-1 interaction. Although the rate of Rb synthesis declines in postmitotic cells, as suggested by In vivo metabolic labeling of the Rb protein, the level of the Rb transcript remains
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1996
It is a characteristic of the central nervous system of higher eukaryotes that neurons, after an ... more It is a characteristic of the central nervous system of higher eukaryotes that neurons, after an initial proliferation phase, remain postmitotic for their whole life span. In the developing quail neuroretina, most retinoblasts become postmitotic after 7-8 days of incubation. They also cease to express cdc2, which is presumably necessary to allow retinoblasts to definitively leave the cell cycle. The molecular mechanisms monitoring cdc2 expression during differentiation remain partly understood. To further study the control of cdc2 transcription in avian cells, we have cloned the quail cdc2 promoter. Two functional regulatory elements have been characterized. One of them contains an E2F-binding site. Human E2F-1 was found to transactivate the quail cdc2 promoter very efficiently in avian and human cells. Gel retardation experiments are presented, suggesting that E2F, in association with different partners, is a major regulatory of cdc2 transcription during the development of the neur...
Biology of the Cell, 1992
unchanged. This suggests that regulation of Rb expression during retina development occurs throug... more unchanged. This suggests that regulation of Rb expression during retina development occurs through posttranscriptional mechanisms. Retinoblastoma (Rb) protein has been implicated in the control of cell proliferation and malignant transformation in different cell types. To analyze ifs role as a promoter of cell growth arrest during development, we have studied the temporal pattern of Rb expression and its association with E2F-1 during embryogenesis of the quail neuroretina. During development of this neural organ, moat cells stop dividing and begin to differentiate at embryonic days E6 and E7, as indicated by the decline of cyclindependent kinase cdk2 and by an increased level of
594 Identification of novel EZH2 inhibitor scaffolds
European Journal of Cancer, 2014
After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukar... more After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukaryotes remain postmitotic during their entire lifespan. This requires that a very stringent control be exerted on the cell division apparatus, whose molecular mechanisms remain quite elusive. Here we have used quail neuroretina as a model to study the control of cell division in the developing CNS. In vertebrates, embryonic neuroretinal cells (NR cells) stop their proliferation at different times depending on the cell type. Most NR cells in the quail embryo become post mitotic between E7 and Ea. To acquire a better understanding of the molecular events leading to quiescence in NR cells, we have analyzed the expression of cdc2and of two activators of p34cdc2:cyclin A and cyclin 82 inthe developing neuroretina. We report that these three proteins are down regulated between E7 and E9, suggesting that a common mechanism could block their transcription in differentiating neurons. We also repor...
Journal of Biological Chemistry, 2001
Pulling Strings Below the Surface: Hormone Receptor Signaling Through Inhibition of Protein Tyrosine Phosphatases
Endocrine, 2001
Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important r... more Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important role as therapeutic agents. Most hormone receptors signal through a mechanism that involves phosphorylation of the receptor's tyrosine residues. At any given moment, the receptor's phosphorylation state depends on the balance of kinase and phosphatase activities. Recent findings point to the exciting possibility that receptor signaling can be regulated by inhibition of protein tyrosine phosphatases (PTPs) that specifically hydrolyze receptor tyrosine-phosphates, or their immediate downstream effectors. This strategy has now been firmly validated for the insulin receptor and PTP1B; inhibiting PTP1B activity results in stimulation of the insulin receptor and signaling, even in the absence of insulin. This and similar findings suggest that PTP inhibitors have potential as hormone mimetics. In the present review, we outline this new paradigm for therapeutic regulation of the insulin receptor and discuss evidence that hints at other specific receptor-PTP pairs.
Oncogene, 1999
cdc2 gene expression is under the control of multiple factors. Although E2F/DP proteins have been... more cdc2 gene expression is under the control of multiple factors. Although E2F/DP proteins have been reported to play a central role, they cannot account for all aspects of the ®ne modulation of cdc2 gene expression during cell cycle and embryonic development. To characterize the transcription factors that control cdc2 gene expression during nerve cell dierentiation in avians, we have previously cloned the quail cdc2 gene promoter region. We had identi®ed an octamer (CAGGTGGC) containing an E-box, which has important activity in regulating cdc2 transcription. Using in vivo genomic footprinting experiments, we show here that this motif, currently named IG, is the target of binding proteins at dierent stages of neuroretina development, con®rming its importance as a regulatory response element for cdc2 gene expression. A subset of Helix ± Loop ± Helix family of transcription factors, known as Upstream Stimulatory Factors (USFs) speci®cally bind to this sequence as dimers. Moreover, our results indicate that USFs transactivate the promoter of cdc2 via the IG motif. These data may help to better understand the mechanisms that control cell division in dierentiating nerve cells.
Journal of Biological Chemistry, 2003
Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently... more Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and K m determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.
Biochemical and Biophysical Research Communications, 2005
Sap-1/PTPRH, a receptor protein tyrosine phosphatase (RPTP), is a ubiquitously expressed enzyme t... more Sap-1/PTPRH, a receptor protein tyrosine phosphatase (RPTP), is a ubiquitously expressed enzyme that is upregulated in human gastrointestinal cancers. Using both chemical cross-linkers and co-immunoprecipitation we show that overexpressed full-length Sap-1 is present as a stable homodimer. Unlike a number of adhesion RPTPs which have tandem catalytic domains that are involved in dimerization, Sap-1 has a single catalytic domain, and we show that this domain is not required for Sap-1 dimerization, which is mediated instead by the large extracellular and transmembrane domains. Exposing cells that express the receptor to a reducing environment reversibly disrupts the Sap-1 dimer, suggesting that cysteine bonds play a role in dimer formation/stabilization. The switch between Sap-1 dimers and monomers is accompanied by an increase in catalytic activity as judged by its capacity to dephosphorylate and activate c-src, which we identify as a novel substrate for this phosphatase.
Journal of Biological Chemistry, 1999
WW domains can be divided into three groups based on their binding specificity. By random mutagen... more WW domains can be divided into three groups based on their binding specificity. By random mutagenesis, we switched the specificity of the Yes-associated protein (YAP) WW1 domain, a Group I WW domain, to that of the FE65 WW domain, which belongs to Group II. We showed that a single mutation, leucine 190 (B5) to tryptophan, is required to switch from Group I to Group II. Although this single substitution in YAP WW1 domain is sufficient to precipitate the two protein isoforms of Mena, an in vivo ligand of FE65, we showed that an additional substitution, histidine 192 (B7) to glycine, significantly increased the ability of YAP to mimic FE65. This double mutant (L190W/H192G) precipitates eight of the nine protein bands that FE65 pulls down from rat brain protein lysates. Based on both our data and a sequence comparison between Group I and Group II WW domains, we propose that a block of three consecutive aromatic amino acids within the second -sheet of the domain is required, but not always sufficient, for a WW domain to belong to Group II. These data deepen our understanding of WW domain binding specificity and provide a basis for the rational design of modified WW domains with potential therapeutic applications.
Journal of Biological Chemistry, 2000
RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical a... more RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation. RSP5 (reversion of Spt phenotype) is an essential gene in the yeast Saccharomyces cerevisiae. The gene was originally isolated in a screen for suppressors of SPT3 mutations (1). SPT3 is part of the multicomponent protein named SAGA (Spt-Ada-Gcn5 acetyltransferase), which has been implicated in at least two facets of RNA pol II 1 activity. These include the function of
Abstract 99: Identification of G9a inhibitors by AlphaLisa™ technology and hit confirmation using MT-Glo™
Cancer Research, 2015
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Epigenetic proce... more Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Epigenetic processes control gene expression and play a major role in cell proliferation, survival and differentiation. Dysregulation of epigenetic mechanisms may lead to alterations in gene expression, resulting in cellular transformation and malignant outgrowth. Epigenetic enzymes are recognized as promising therapeutic targets for the treatment of cancer. Inventiva has several research programs in epigenetics, with specific focus on histone lysine methyltransferases (HKMTs). Emerging evidence suggests that G9a, a HKMT, able to mono and dimethylate H3K9 leading to the inactivation of tumor suppressor genes, plays a predominant role in lung and ovarian cancers. To identify novel G9a inhibitors, we used an AlphaLisa assay, with a histone H3-derived peptide and a specific antibody against H3K9me2. The assay, in 384-well format, displayed a robust Z-factor (0.84) with a signal to noise ratio around 800. We screened Inventiva's proprietary compound library using this assay. Three hundred thirty-six hits inhibited G9a by more than 60% (Hit rate: 0.14%). Despite the advantages offered by AlphaLisa technology, such as high S/N ratios and straightforward automation, this technology has been reported to generate a high number of false positives. In order to confirm the activity of our hits, we used a different technology, the methyltransferase Glo (MT-Glo) assay, which measures the reaction product SAH by luminescence read-out. Out of the 336 identified compounds, 75 were confirmed by this orthogonal screening (Hit confirmation rate: 22%). Additional compound characterization is ongoing to allow the selection of the best hit series for chemical optimization. In conclusion, the AlphaLisa assay, coupled with MT-Glo assay allowed the identification and confirmation of potent hits against G9a, as chemical starting points for our hit-to-lead optimization program. Citation Format: Claudia Fromond, Xavier Espanel, Laurent Chene, Philippe Masson, Benaissa Boubia, Christian Montalbetti, Pierre Broqua. Identification of G9a inhibitors by AlphaLisa™ technology and hit confirmation using MT-Glo™. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 99. doi:10.1158/1538-7445.AM2015-99
Mecanismes transcriptionnels impliques dans le controle de la proliferation cellulaire au cours du developpement de la neuroretine aviaire
Afin de comprendre les mecanismes moleculaires lies a l'arret des divisions et au maintien de... more Afin de comprendre les mecanismes moleculaires lies a l'arret des divisions et au maintien de la quiescence des cellules neuronales, nous avons analyse l'expression du gene cdc2 au cours du developpement de la neuroretine aviaire. Nous avons montre que l'expression de ce gene est reprimee entre les jours 6 et 8 du developpement embryonnaire (e8). L'analyse fonctionnelle du promoteur cdc2 aviaire revele que son activite est dependante de la presence des motifs ig et e2f. Le facteur de transcription e2f-1 transactive le promoteur cdc2 via l'element de reponse e2f, alors que la proteine prb inhibe cette transactivation. L'accumulation de la forme antiproliferative de prb au cours de la neurogenese, suggere qu'elle serait responsable de l'inhibition de l'expression de cdc2 observee entre e6 et e8. A partir de e8, il y a disparition des complexes proteiques se fixant sur le motif e2f. Cette disparition resulte sans doute de l'arret de l'express...
Abstract 4524: A rational approach for the discovery of inhibitors of NSD2 for the treatment of multiple myeloma
Cancer Research, 2016
The WW Domain of Dystrophin Requires EF-Hands Region to Interact with �-Dystroglycan
Biol Chem, 1999
Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytosk... more Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytoskeleton and the extracellular matrix. Perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein beta-dystroglycan, may lead to muscular dystrophy. Previously, the cysteine-rich region and first half of the carboxy-terminal domain of dystrophin were shown to interact with beta-dystroglycan through a stretch of fifteen amino acids at the carboxy-terminus of beta-dystroglycan. This region of dystrophin implicated in binding beta-dystroglycan contains four modular protein domains: a WW domain, two putative Ca2+-binding EF-hand motifs, and a putative zinc finger ZZ domain. The WW domain is a globular domain of 38-40 amino acids with two highly conserved tryptophan residues spaced 20-22 amino acids apart. A subset of WW domains was shown to bind ligands that contain a Pro-Pro-x-Tyr core motif (where x is any amino acid). Here we elucidate the role of the WW domain of dystrophin and surrounding sequence in binding beta-dystroglycan. We show that the WW domain of dystrophin along with the EF-hand motifs binds to the carboxy-terminus of beta-dystroglycan. Through site-specific mutagenesis and in vitro binding assays, we demonstrate that binding of dystrophin to the carboxy-terminus of beta-dystroglycan occurs via a beta-dystroglycan Pro-Pro-x-Tyr core motif. Targeted mutagenesis of conserved WW domain residues reveals that the dystrophin/beta-dystroglycan interaction occurs primarily through the WW domain of dystrophin. Precise mapping of this interaction could aid in therapeutic design.
Mecanismes transcriptionnels impliques dans le controle de la proliferation cellulaire au cours du developpement de la neuroretine aviaire
Http Www Theses Fr, 1997
Oncogene, 1998
E2F-1 is the prototype of a family of transcription factors playing a central role in the control... more E2F-1 is the prototype of a family of transcription factors playing a central role in the control of cell proliferation and apoptosis. E2F DNA binding activity is down-regulated during cellular dierentiation, which is correlated with cell division arrest. We report here that the expression of E2F-1 itself is down-regulated in the developing quail neural retina between embryonic days E8-E10. This event occurs just after the massive arrest of the quail neuroretina cell division (E7-E8). To gain further insight into the regulatory mechanisms monitoring E2F-1 expression in dierentiating neurons, we have cloned the quail E2F-1 promoter. In vivo DNA footprintings of this promoter have shown that a number of potential SP-1 and C/EBP response elements are constitutively occupied in the entire quail neuroretina of E5 and E14, whereas the two consensus palindromic E2F binding sites are only protected at E5. This suggests that these E2F elements participate in down-regulation of E2F-1 gene expression during avian neuroretina development. CAT reporter assays have shown that E2F-1 in association with its partner DP-1 transactivates its own promoter, whereas p105 Rb inhibits the E2F-1 promoter. Both E2F-1/DP-1 and p105 Rb require the presence of the E2F binding sites to mediate their eects. However, experiments performed with deletion mutants of the promoter strongly suggest that other regions located upstream of the E2F binding sites also mediate part of the E2F-1 transactivating eect on its own promoter. Altogether, these results suggest that the down-regulation of E2F-1 gene expression in dierentiating neurons could be due, in part, to the E2F/Rb complexes binding to the E2F-1 promoter.
HAL (Le Centre pour la Communication Scientifique Directe), Oct 1, 1998
Retinoblastoma (Rb) protein has been implicated in the control of cell proliferation and malignan... more Retinoblastoma (Rb) protein has been implicated in the control of cell proliferation and malignant transformation in different cell types. To analyze ifs role as a promoter of cell growth arrest during development, we have studied the temporal pattern of Rb expression and its association with E2F-1 during embryogenesis of the quail neuroretina. During development of this neural organ, moat cells stop dividing and begin to differentiate at embryonic days E6 and E7, as indicated by the decline of cyclindependent kinase cdk2 and by an increased level of cdk5. At this stage, we observed a shift of hyperphosphorylated Rb protein to Its hypophosphorylated form as well as a decrease of the total level of Rb. The Rb-related protein, p107, is also progressively down-regulated from the E7 stage onwards. P130 levels, on the other hand, actually increase. Moreover, cell cycle exit at E6-E7 is characterized by a sudden and transient rise of the E2F-1/RB complex followed by the appearance of the E2F-4/p130 complex starting at E8. Conversely, expression of adenovirus EIA protein in E6 neuroretina cells leads to a dissociation of E2F-1/Rb complex and suppression of cell growth arrest and differentiation. This suggests that cell cycle exit and re-entry may depend on Rb/E2F-1 interaction. Although the rate of Rb synthesis declines in postmitotic cells, as suggested by In vivo metabolic labeling of the Rb protein, the level of the Rb transcript remains
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1996
It is a characteristic of the central nervous system of higher eukaryotes that neurons, after an ... more It is a characteristic of the central nervous system of higher eukaryotes that neurons, after an initial proliferation phase, remain postmitotic for their whole life span. In the developing quail neuroretina, most retinoblasts become postmitotic after 7-8 days of incubation. They also cease to express cdc2, which is presumably necessary to allow retinoblasts to definitively leave the cell cycle. The molecular mechanisms monitoring cdc2 expression during differentiation remain partly understood. To further study the control of cdc2 transcription in avian cells, we have cloned the quail cdc2 promoter. Two functional regulatory elements have been characterized. One of them contains an E2F-binding site. Human E2F-1 was found to transactivate the quail cdc2 promoter very efficiently in avian and human cells. Gel retardation experiments are presented, suggesting that E2F, in association with different partners, is a major regulatory of cdc2 transcription during the development of the neur...
Biology of the Cell, 1992
unchanged. This suggests that regulation of Rb expression during retina development occurs throug... more unchanged. This suggests that regulation of Rb expression during retina development occurs through posttranscriptional mechanisms. Retinoblastoma (Rb) protein has been implicated in the control of cell proliferation and malignant transformation in different cell types. To analyze ifs role as a promoter of cell growth arrest during development, we have studied the temporal pattern of Rb expression and its association with E2F-1 during embryogenesis of the quail neuroretina. During development of this neural organ, moat cells stop dividing and begin to differentiate at embryonic days E6 and E7, as indicated by the decline of cyclindependent kinase cdk2 and by an increased level of
594 Identification of novel EZH2 inhibitor scaffolds
European Journal of Cancer, 2014
After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukar... more After an initial proliferation phase, neurons of the central nervous system (CNS) of higher eukaryotes remain postmitotic during their entire lifespan. This requires that a very stringent control be exerted on the cell division apparatus, whose molecular mechanisms remain quite elusive. Here we have used quail neuroretina as a model to study the control of cell division in the developing CNS. In vertebrates, embryonic neuroretinal cells (NR cells) stop their proliferation at different times depending on the cell type. Most NR cells in the quail embryo become post mitotic between E7 and Ea. To acquire a better understanding of the molecular events leading to quiescence in NR cells, we have analyzed the expression of cdc2and of two activators of p34cdc2:cyclin A and cyclin 82 inthe developing neuroretina. We report that these three proteins are down regulated between E7 and E9, suggesting that a common mechanism could block their transcription in differentiating neurons. We also repor...
Journal of Biological Chemistry, 2001
Pulling Strings Below the Surface: Hormone Receptor Signaling Through Inhibition of Protein Tyrosine Phosphatases
Endocrine, 2001
Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important r... more Hormones, cytokines, and related proteins (such as soluble hormone receptors) play an important role as therapeutic agents. Most hormone receptors signal through a mechanism that involves phosphorylation of the receptor's tyrosine residues. At any given moment, the receptor's phosphorylation state depends on the balance of kinase and phosphatase activities. Recent findings point to the exciting possibility that receptor signaling can be regulated by inhibition of protein tyrosine phosphatases (PTPs) that specifically hydrolyze receptor tyrosine-phosphates, or their immediate downstream effectors. This strategy has now been firmly validated for the insulin receptor and PTP1B; inhibiting PTP1B activity results in stimulation of the insulin receptor and signaling, even in the absence of insulin. This and similar findings suggest that PTP inhibitors have potential as hormone mimetics. In the present review, we outline this new paradigm for therapeutic regulation of the insulin receptor and discuss evidence that hints at other specific receptor-PTP pairs.
Oncogene, 1999
cdc2 gene expression is under the control of multiple factors. Although E2F/DP proteins have been... more cdc2 gene expression is under the control of multiple factors. Although E2F/DP proteins have been reported to play a central role, they cannot account for all aspects of the ®ne modulation of cdc2 gene expression during cell cycle and embryonic development. To characterize the transcription factors that control cdc2 gene expression during nerve cell dierentiation in avians, we have previously cloned the quail cdc2 gene promoter region. We had identi®ed an octamer (CAGGTGGC) containing an E-box, which has important activity in regulating cdc2 transcription. Using in vivo genomic footprinting experiments, we show here that this motif, currently named IG, is the target of binding proteins at dierent stages of neuroretina development, con®rming its importance as a regulatory response element for cdc2 gene expression. A subset of Helix ± Loop ± Helix family of transcription factors, known as Upstream Stimulatory Factors (USFs) speci®cally bind to this sequence as dimers. Moreover, our results indicate that USFs transactivate the promoter of cdc2 via the IG motif. These data may help to better understand the mechanisms that control cell division in dierentiating nerve cells.
Journal of Biological Chemistry, 2003
Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently... more Protein tyrosine phosphatases (PTPs) play important, highly dynamic roles in signaling. Currently about 90 different PTP genes have been described. The enzymes are highly regulated at all levels of expression, and it is becoming increasingly clear that substrate specificity of the PTP catalytic domains proper contributes considerably to PTP functionality. To investigate PTP substrate selectivity, we have set up a procedure to generate phage libraries that presents randomized, phosphotyrosine-containing peptides. Phages that expressed suitable substrates were selected by immobilized, substrate-trapping GST-PTP fusion proteins. After multiple rounds of selection, positive clones were confirmed by SPOT analysis, dephosphorylation by wild-type enzyme, and K m determinations. We have identified distinct consensus substrate motifs for PTP1B, Sap-1, PTP-, SHP1, and SHP2. Our results confirm substrate specificity for individual PTPs at the peptide level. Such consensus sequences may be useful both for identifying potential PTP substrates and for the development of peptidomimetic inhibitors.
Biochemical and Biophysical Research Communications, 2005
Sap-1/PTPRH, a receptor protein tyrosine phosphatase (RPTP), is a ubiquitously expressed enzyme t... more Sap-1/PTPRH, a receptor protein tyrosine phosphatase (RPTP), is a ubiquitously expressed enzyme that is upregulated in human gastrointestinal cancers. Using both chemical cross-linkers and co-immunoprecipitation we show that overexpressed full-length Sap-1 is present as a stable homodimer. Unlike a number of adhesion RPTPs which have tandem catalytic domains that are involved in dimerization, Sap-1 has a single catalytic domain, and we show that this domain is not required for Sap-1 dimerization, which is mediated instead by the large extracellular and transmembrane domains. Exposing cells that express the receptor to a reducing environment reversibly disrupts the Sap-1 dimer, suggesting that cysteine bonds play a role in dimer formation/stabilization. The switch between Sap-1 dimers and monomers is accompanied by an increase in catalytic activity as judged by its capacity to dephosphorylate and activate c-src, which we identify as a novel substrate for this phosphatase.
Journal of Biological Chemistry, 1999
WW domains can be divided into three groups based on their binding specificity. By random mutagen... more WW domains can be divided into three groups based on their binding specificity. By random mutagenesis, we switched the specificity of the Yes-associated protein (YAP) WW1 domain, a Group I WW domain, to that of the FE65 WW domain, which belongs to Group II. We showed that a single mutation, leucine 190 (B5) to tryptophan, is required to switch from Group I to Group II. Although this single substitution in YAP WW1 domain is sufficient to precipitate the two protein isoforms of Mena, an in vivo ligand of FE65, we showed that an additional substitution, histidine 192 (B7) to glycine, significantly increased the ability of YAP to mimic FE65. This double mutant (L190W/H192G) precipitates eight of the nine protein bands that FE65 pulls down from rat brain protein lysates. Based on both our data and a sequence comparison between Group I and Group II WW domains, we propose that a block of three consecutive aromatic amino acids within the second -sheet of the domain is required, but not always sufficient, for a WW domain to belong to Group II. These data deepen our understanding of WW domain binding specificity and provide a basis for the rational design of modified WW domains with potential therapeutic applications.
Journal of Biological Chemistry, 2000
RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical a... more RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation. RSP5 (reversion of Spt phenotype) is an essential gene in the yeast Saccharomyces cerevisiae. The gene was originally isolated in a screen for suppressors of SPT3 mutations (1). SPT3 is part of the multicomponent protein named SAGA (Spt-Ada-Gcn5 acetyltransferase), which has been implicated in at least two facets of RNA pol II 1 activity. These include the function of
Abstract 99: Identification of G9a inhibitors by AlphaLisa™ technology and hit confirmation using MT-Glo™
Cancer Research, 2015
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Epigenetic proce... more Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Epigenetic processes control gene expression and play a major role in cell proliferation, survival and differentiation. Dysregulation of epigenetic mechanisms may lead to alterations in gene expression, resulting in cellular transformation and malignant outgrowth. Epigenetic enzymes are recognized as promising therapeutic targets for the treatment of cancer. Inventiva has several research programs in epigenetics, with specific focus on histone lysine methyltransferases (HKMTs). Emerging evidence suggests that G9a, a HKMT, able to mono and dimethylate H3K9 leading to the inactivation of tumor suppressor genes, plays a predominant role in lung and ovarian cancers. To identify novel G9a inhibitors, we used an AlphaLisa assay, with a histone H3-derived peptide and a specific antibody against H3K9me2. The assay, in 384-well format, displayed a robust Z-factor (0.84) with a signal to noise ratio around 800. We screened Inventiva's proprietary compound library using this assay. Three hundred thirty-six hits inhibited G9a by more than 60% (Hit rate: 0.14%). Despite the advantages offered by AlphaLisa technology, such as high S/N ratios and straightforward automation, this technology has been reported to generate a high number of false positives. In order to confirm the activity of our hits, we used a different technology, the methyltransferase Glo (MT-Glo) assay, which measures the reaction product SAH by luminescence read-out. Out of the 336 identified compounds, 75 were confirmed by this orthogonal screening (Hit confirmation rate: 22%). Additional compound characterization is ongoing to allow the selection of the best hit series for chemical optimization. In conclusion, the AlphaLisa assay, coupled with MT-Glo assay allowed the identification and confirmation of potent hits against G9a, as chemical starting points for our hit-to-lead optimization program. Citation Format: Claudia Fromond, Xavier Espanel, Laurent Chene, Philippe Masson, Benaissa Boubia, Christian Montalbetti, Pierre Broqua. Identification of G9a inhibitors by AlphaLisa™ technology and hit confirmation using MT-Glo™. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 99. doi:10.1158/1538-7445.AM2015-99
Mecanismes transcriptionnels impliques dans le controle de la proliferation cellulaire au cours du developpement de la neuroretine aviaire
Afin de comprendre les mecanismes moleculaires lies a l'arret des divisions et au maintien de... more Afin de comprendre les mecanismes moleculaires lies a l'arret des divisions et au maintien de la quiescence des cellules neuronales, nous avons analyse l'expression du gene cdc2 au cours du developpement de la neuroretine aviaire. Nous avons montre que l'expression de ce gene est reprimee entre les jours 6 et 8 du developpement embryonnaire (e8). L'analyse fonctionnelle du promoteur cdc2 aviaire revele que son activite est dependante de la presence des motifs ig et e2f. Le facteur de transcription e2f-1 transactive le promoteur cdc2 via l'element de reponse e2f, alors que la proteine prb inhibe cette transactivation. L'accumulation de la forme antiproliferative de prb au cours de la neurogenese, suggere qu'elle serait responsable de l'inhibition de l'expression de cdc2 observee entre e6 et e8. A partir de e8, il y a disparition des complexes proteiques se fixant sur le motif e2f. Cette disparition resulte sans doute de l'arret de l'express...
Abstract 4524: A rational approach for the discovery of inhibitors of NSD2 for the treatment of multiple myeloma
Cancer Research, 2016
The WW Domain of Dystrophin Requires EF-Hands Region to Interact with �-Dystroglycan
Biol Chem, 1999
Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytosk... more Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytoskeleton and the extracellular matrix. Perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein beta-dystroglycan, may lead to muscular dystrophy. Previously, the cysteine-rich region and first half of the carboxy-terminal domain of dystrophin were shown to interact with beta-dystroglycan through a stretch of fifteen amino acids at the carboxy-terminus of beta-dystroglycan. This region of dystrophin implicated in binding beta-dystroglycan contains four modular protein domains: a WW domain, two putative Ca2+-binding EF-hand motifs, and a putative zinc finger ZZ domain. The WW domain is a globular domain of 38-40 amino acids with two highly conserved tryptophan residues spaced 20-22 amino acids apart. A subset of WW domains was shown to bind ligands that contain a Pro-Pro-x-Tyr core motif (where x is any amino acid). Here we elucidate the role of the WW domain of dystrophin and surrounding sequence in binding beta-dystroglycan. We show that the WW domain of dystrophin along with the EF-hand motifs binds to the carboxy-terminus of beta-dystroglycan. Through site-specific mutagenesis and in vitro binding assays, we demonstrate that binding of dystrophin to the carboxy-terminus of beta-dystroglycan occurs via a beta-dystroglycan Pro-Pro-x-Tyr core motif. Targeted mutagenesis of conserved WW domain residues reveals that the dystrophin/beta-dystroglycan interaction occurs primarily through the WW domain of dystrophin. Precise mapping of this interaction could aid in therapeutic design.
Mecanismes transcriptionnels impliques dans le controle de la proliferation cellulaire au cours du developpement de la neuroretine aviaire
Http Www Theses Fr, 1997
Oncogene, 1998
E2F-1 is the prototype of a family of transcription factors playing a central role in the control... more E2F-1 is the prototype of a family of transcription factors playing a central role in the control of cell proliferation and apoptosis. E2F DNA binding activity is down-regulated during cellular dierentiation, which is correlated with cell division arrest. We report here that the expression of E2F-1 itself is down-regulated in the developing quail neural retina between embryonic days E8-E10. This event occurs just after the massive arrest of the quail neuroretina cell division (E7-E8). To gain further insight into the regulatory mechanisms monitoring E2F-1 expression in dierentiating neurons, we have cloned the quail E2F-1 promoter. In vivo DNA footprintings of this promoter have shown that a number of potential SP-1 and C/EBP response elements are constitutively occupied in the entire quail neuroretina of E5 and E14, whereas the two consensus palindromic E2F binding sites are only protected at E5. This suggests that these E2F elements participate in down-regulation of E2F-1 gene expression during avian neuroretina development. CAT reporter assays have shown that E2F-1 in association with its partner DP-1 transactivates its own promoter, whereas p105 Rb inhibits the E2F-1 promoter. Both E2F-1/DP-1 and p105 Rb require the presence of the E2F binding sites to mediate their eects. However, experiments performed with deletion mutants of the promoter strongly suggest that other regions located upstream of the E2F binding sites also mediate part of the E2F-1 transactivating eect on its own promoter. Altogether, these results suggest that the down-regulation of E2F-1 gene expression in dierentiating neurons could be due, in part, to the E2F/Rb complexes binding to the E2F-1 promoter.