DESCRIPTIF DU TRAVAIL DE RECHERCHE
The cell signaling group investigates cell signaling pathways and new regulatory mechanisms involved in autophagy, apoptosis and in a genetic disease affecting the nervous system : neurofibromatosis type 1 (NF1). Our scientific strategy is to develop research axes in two directions : fundamental research dedicated to the understanding of molecular mechanisms and applied research dedicated to the identification and development of new therapeutic targets and diagnostic tools. Our domains of expertise are cell culturing, molecular biology, quantitative PCR, protein-protein interaction studies and biochemistry. We collaborate with teams specialized in 3D modeling, organic chemistry and with clinicians.
Autophagy and apoptosis :
GALIG, a human gene embedded in the Galectin-3 gene has been initially described as a proapoptotic gene. It encodes two unrelated proteins, Mitogaligin and Cytogaligin. Proapoptotic properties have been established in relation with Mitogaligin which destabilizes mitochondrial membranes through specific interaction with cardiolipin, a mitochondrial phospholipid. GALIG is underexpressed in bone marrow of patients with Acute Myeloid Leukemia (AML). Mitogaligin and Cytogaligin have no homology with any other known proteins and their mechanism of action is still poorly understood. Recently, a partial Cytogaligin interactome has been established and reveals that GALIG could be associated with several additional biological functions related to the protein degradation pathways. Indeed, Cytogaligin interacts with several proteins related to autophagy and UPS (Ubiquitin-proteasome system). It interacts also with alpha-synuclein, a major protein in Parkinson’s disease. Our research focuses on the functional consequences of theses interactions in order to determine the potential role of Cytogaligin during autophagy and apoptosis.
We also develop a translational project, in collaboration with several services from the Hospital of Orléans. The objective is to investigate GALIG gene expression and others genes in different pathologies and more specifically in acute myeloid leukemia (AML), Parkinson’s disease (PD) and human immunodeficiency (HIV) infection. Preliminary data obtained from AML patients support the potential role of GALIG during differentiation of myeloid cells which is a critical point in the treatment and diagnosis of the disease. In addition, the level of GALIG expression returns to a normal one following chemotherapy treatments. Also, HIV patients exhibit an abnormal level of GALIG gene expression. These results encourage us to further investigate the role of GALIG in AML and HIV infection. Regarding Parkinson’s disease, preliminary data obtained from a small cohort of patients, revealed a clear disturbance of expression of several autophagy genes in peripheral blood. Further investigation are conducted to determine whether such deregulated genes may be considered as a molecular marker for prognostic and diagnostic.
Neurofibromatosis type 1 :
Neurofibromatosis type 1 (NF1) is a very common genetic disease. NF1 phenotype is highly variable, however the hallmark of NF1 is the development of nerve tumors with an increased risk of malignancies, and neurological disorders. Currently, NF1 treatments remain poorly efficient, and identifying new therapeutic targets to treat this disease is a challenge for the forthcoming years.
NF1 is due to mutations within the NF1 tumor suppressor gene, which encodes for neurofibromin, Nf1, a huge protein. We performed a two-hybrid screening and identified new partners of Nf1. We decided to focus on three of them, LINGO-1, LARP6, and LIMK2. We are also working on 5-HT6 receptor, which was identified as a new partner of Nf1 by a proteomic approach developed by the team of P.Marin (IGF, Montpellier). 5-HT6 receptor is a serotonin receptor which is a promising target for the treatment of cognitive deficits. Its constitutive activity on cAMP is regulated by Nf1. LINGO-1 plays a key role in different processes of the central nervous system (neuronal growth and survival, myelinisation), we are studying its functional interaction with Nf1. LARP6 regulates type I collagen mRNA translation, we showed that Nf1 disturbs the interaction between LARP6 and type I collagen mRNA. LIMK2 is a kinase involved in cytoskeleton remodeling, especially actin cytoskeleton via Rho/ROCK/LIMK2/cofilin pathway. We showed that Nf1 negatively regulates this pathway by preventing LIMK2 activation by ROCK. We are also working on the three human isoforms of LIMK2, and we showed that one of them is involved in mental deficiency.
On another hand, we are part of a collaborative network with bioanalysts and chemists, to develop new inhibitors of LIM kinases (LIMK1 and LIMK2). Besides the connexion between NF1 and LIMK2, LIM kinases are misregulated in many cancers. They constitute new attractive therapeutic targets. In a previous similar collaborative project, we have developped new inhibitors of PI3K and mTOR, the most efficient molecules are currently under preclinical trial.
and is connected to diverse signaling pathways
Collaborations on autophagy and apoptosis :
Centre Hospitalier Régional d’Orléans :
Service de Neurologie, Drs Canan OZSANCAK et Pascal AUZOU
Services d’Oncologie Médicale et Hématologie Clinique (Pôle Médecines interventionnelles), Drs Michèle SCHOENWALD et Diana CARP.
Unité de Génétique Dr Sylvain BRIAULT et Thomas GUERY
Unité d’Hématologie (Pôle Biopathologie), Dr Eric LEGAC.
Service des Maladies Infectieuses et Tropicales, Drs Laurent HOCQUELOUX et Thierry PRAZUCK.
Centre d’études d’agents Pathogènes et Biotechnologies pour la Santé - CNRS UMR 5236,
Université de Franche-Comté, Besançon. Unité Estrogènes, expression génique et pathologies du
système nerveux central (E2SNC), Pr Michaël BOYER-GUITTAUT
Groupe hospitalier l’Archet, Nice.
Service d’Hématologie Clinique, Dr Pierre-Simon ROHRLICH
Collaborations on Neurofibromatosis type 1 :
CBM, Orléans :
Equipe « Aspects moléculaires du vivant »
Groupe thématique : « Réparation de l’ADN : structure, fonction et dynamique » Dr. F. Culard
Groupe thématique : « Spectrométrie de masse fonctionnelle des métastases » Dr. M. Cadene
ICOA, Université d’Orléans :
« Synthèse hétérocyclique et chimie thérapeutique » Pr. S. Routier, Dr. K. Plé, Dr. F. Buron
« Bioinformatique structurale et chemoinformatique » Pr. P. Bonnet, Dr. S. Aci
« Extraction, analyse de molécules bioactives » Dr. R. Néhmé
Chimie Organique et Chimie thérapeutique, Université de Nantes : Pr. J.M. Robert
IMVC, Université de Rennes 1 : Pr. JP Bazureau
Station Biologique de Roscoff
« Plateforme de criblage KISSF » Dr. S. Ruchaud
INSERM U1069, Faculté de Médecine Tours : Dr. M.L Jourdan
INSERM U930, Faculté de Médecine Tours :
« Neurogénétique et neurométabolomique » Pr. C. Andrès, Dr. P. Vourc’h
IGF, Département Neurosciences, Montpellier : Dr. P. Marin
Division of Cancer and Genetics, Cardiff University, Grand Bretagne : Pr. M. Upadhyaya
KeyObs, Orléans : F. Trovero
- Deraredj Nadim W., Chaumont-Dubel S., Madouri F., Cobret L., De Tauzia M.-L., Zajdel P., Bénédetti H., Marin P. and Morisset-Lopez S. (2016) A Physical Interaction between Neurofibromin and Serotonin 5-HT6 Receptor Promotes Receptor Constitutive Activity. Proc Natl Acad Sci U S A, in press
- Saurat T., Buron F., Rodriguès N., de Tauzia M.-L., Colliandre L., Bourg S., Bonnet P., Guillaumet G., Akssira M., Corlu A., Guillouzo C., Berthier P., Rio P., Jourdan ML., Bénédetti H. and Routier S. (2014) Design, Synthesis and Biological Activity of Pyridopyrimidine Scaffolds as Novel PI3K/ mTOR Dual Inhibitors. J. Med. Chem., 57, 613-631.
- Mollet L., Robinet P., Dubois M., Aurouet A., Normand T., Charpentier S., Sureau A., Grandclement C., Garnache-Ottou F., Deconinck E., Brulé F., Rohrlich P.-S. and Legrand A. (2013) Opposing Mcl-1, the GALIG proapoptotic gene is upregulated as neutrophils die and underexpressed in Acute Myeloid Leukemia cells. Molecular Immunology, 56, 123-128.
- Vallée B., Doudeau M., Godin F., Gombault A., Tchalikian A., de Tauzia M.-L., and Bénédetti H. (2012) Nf1 RasGAP Inhibition of LIMK2 Mediates a New Cross- Talk between Ras and Rho Pathways. PLOS One, 7, e47283.
- Godin F., Villette S., Vallée B., Doudeau M., Morisset-Lopez S., Ardourel M., Hevor T., Pichon C. and Bénédetti H. (2012) A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell line. BBRC, 418, 689-694.
- Robinet P., Mollet L., Gonzalez P., Normand T., Charpentier S., Brulé F., Dubois M. and Legrand A. (2010) The mitogaligin protein is addressed to the nucleus via a non classical localization signal. Biochem. Biophys. Res. Commun., 392, 53-57.
- Gonzalez P., Duneau M., Charpentier S., Normand T., Mollet L., Dubois M., and Legrand A. (2007) Destabilization of membranes containing cardiolipin or its precursors by peptides derived from mitogaligin, a cell death protein. Biochemistry, 46, 7374-7382.
Nadim W. D., Simion V., Bénédetti H., Pichon C., Baril P. and Morisset-Lopez S. (2016)
Neurodegenerative and cognitive disorders are multifactorial diseases (i.e., involving neurodevelopmental, genetic, age or environmental factors) that are characterized by an abnormal development affecting neuronal function and integrity. Recently, an increasing number of studies revealed that the dysregulation of microRNAs (miRNAs) may be involved in the etiology of cognitive disorders such as Alzheimer, Parkinson, and Huntington’s diseases, Schizophrenia and Autism spectrum disorders. MiRNAs are a class of small non-coding RNAs that regulate gene expression through a base pairing mechanism with their target mRNAs, thereby inducing translational repression or mRNA degradation. Intriguingly, 70 % of known miRNAs are expressed in the brain while functional studies indicate that they play crucial roles in brain development by regulating key signaling pathways involved in synaptogenesis, neuronal plasticity, neurite outgrowth and memory processes. Further, the relevance of modulating miRNA expression levels to correct neurocognitive disorders is supported by the increasing number of studies demonstrating a link between miRNA dysregulation and the etiology and/or pathophysiology of cognitive dysfunction in several neurologic and neuropsychiatric disorders. This suggests that miRNAs might be used as diagnosis markers and/or could be exploited for therapeutic interventions. In this review, we first briefly summarize the basic molecular events involved in memory, a process inherent to cognitive disease, and then describe the role of miRNAs in neurodevelopment, synaptic plasticity and memory. Secondly, we provide an overview of the impact of miRNA dysregulation in the pathogenesis of different neurocognitive disorders, and lastly discuss the feasibility of miRNA-based therapeutics in the treatment of these cognitive disorders. This last part will be discussed in detail in the following review.
Physical Interaction between Neurofibromin and Serotonin 5-HT6 Receptor Promotes Receptor Constitutive ActivityProc Natl Acad Sci U S A (2016) 113 (43) 12310-12315 - doi : 10.1073/pnas.1600914113
Active G protein-coupled receptor (GPCR) conformations not only are promoted by agonists but also occur in their absence, leading to constitutive activity. Association of GPCRs with intracellular protein partners might be one of the mechanisms underlying GPCR constitutive activity. Here, we show that serotonin 5 hydroxytryptamine 6 (5-HT6) receptor constitutively activates the Gs/adenylyl cyclase pathway in various cell types, including neurons. Constitutive activity is strongly reduced by silencing expression of the Ras-GTPase activating protein (Ras-GAP) neurofibromin, a 5-HT6 receptor partner. Neurofibromin is a multidomain protein encoded by the NF1 gene, the mutation of which causes Neurofibromatosis type 1 (NF1), a genetic disorder characterized by multiple benign and malignant nervous system tumors and cognitive deficits. Disrupting association of 5-HT6 receptor with neurofibromin Pleckstrin Homology (PH) domain also inhibits receptor constitutive activity, and PH domain expression rescues 5-HT6 receptor-operated cAMP signaling in neurofibromin-deficient cells. Furthermore, PH domains carrying mutations identified in NF1 patients that prevent interaction with the 5-HT6 receptor fail to rescue receptor constitutive activity in neurofibromin-depleted cells. Further supporting a role of neurofibromin in agonist-independent Gs signaling elicited by native receptors, the phosphorylation of cAMP-responsive element-binding protein (CREB) is strongly decreased in prefrontal cortex of Nf1+/− mice compared with WT mice. Moreover, systemic administration of a 5-HT6 receptor inverse agonist reduces CREB phosphorylation in prefrontal cortex of WT mice but not Nf1+/− mice. Collectively, these findings suggest that disrupting 5-HT6 receptor–neurofibromin interaction prevents agonist-independent 5-HT6 receptor-operated cAMP signaling in prefrontal cortex, an effect that might underlie neuronal abnormalities in NF1 patients.
Tastet J., Decalonne L., Marouillat S., Malvy J., Thepault R. A., Toutain A., Paubel A., Tabagh R., Benedetti H., Laumonnier F., Barthelemy C., Bonnet-Brilhault F., Andres C. R. and Vourc’h P. (2015)
Many genes are now thought to confer susceptibility to autism. Despite the fact that this neuropsychiatric disease appears to be related to several different causes, common cellular and molecular pathways have emerged and point to synaptic dysfunction or cellular growth. Several studies have indicated the importance of the ubiquitin pathway in synaptic function and the aetiology of autism. Here, we focused on the ring finger protein 135 (RNF135) gene, encoding an E3 ubiquitin ligase expressed in the cortex and cerebellum, and located in the NF1 gene locus in 17q11.2, a region linked to autism. We carried out a genetic analysis of the coding sequence of RFN135 in a French cohort of patients with autism and observed a significantly increased frequency of genotypes carrying the rare allele of the rs111902263 (p.R115K) missense variant in patients (P=0.0019, odds ratio : 4.23, 95% confidence interval : 1.87-9.57). Particularly, three unrelated patients showed a homozygous genotype for K115, a situation not observed in the 1812 control individuals. Further cellular and molecular studies are required to elucidate the role of this gene and the variant K115 in brain development and neuronal function.
We finally present the existence of a cofilin-independent pathway also involved in neuronal function. A better understanding of the differences between both LIMKs and of the precise molecular mechanisms involved in their mode of action and regulation is now required to improve our understanding of the physiopathology of the neuronal diseases associated with LIMKs.
Background and Purpose The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. Experimental Approach LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein–protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. Key Results LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. Conclusions and Implications Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery.
Bruel A., Bénéteau R. Chabanne M., Lozach O. Le Guevel R., Ravache M., Bénédetti H., Meijer L., Logé C., Robert J.-M. (2014)
New pyridazino[4,5-b]indol-4-ones and pyridazin-3(2H)-one analogs were synthesized and their inhibitory activities against DYRK1A, CDK5/p25, GSK3α/β and p110-α isoform of PI3K evaluated using harmine as reference. Both furan-2-yl 10 and pyridin-4-yl 19 from the two different series, exhibited submicromolar IC50 against DYRK1A with no activities against the three other kinases. In addition, compound 10 exhibited antiproliferative activities in the Huh-7, Caco2 and MDA-MB-231 cell lines.
Microwave Assisted Organic Synthesis (MAOS) of New Dispacamide A Derivatives Bearing a Thiazolinone Platform, Biological Assays on Inhibition of Protein Kinases and Cell EffectsCurrent Microwave Chemistry (2014) 1 (1) 33-40 - doi : 10.2174/22133356114019990002
A series of (5Z)-5-ylidene-thiazolidine-4-one derivatives bearing the N-(4,5-dihalogenopyrrol-2-yl)carbamoyl fragment of Dispacamide A was prepared through a newly developed approach using a solution phase protocol assisted by microwave irradiation. These new compounds were synthesized in good yields (10-98%) by sulphur/nitrogen displacement or eventually by Knoevenagel condensation in the presence of a base (AcONa) in AcOH solution. The ten synthetic products have been obtained with a Z-geometry about their exocyclic double bond. All these compounds have been evaluated against eight protein kinases and human cell lines.
New in-capillary electrophoretic kinase assays to evaluate inhibitors of the PI3k/Akt/mTOR signaling pathwayAnalytical and Bioanalytical Chemistry (2014) 406 (13) 3743-3754 - doi : 10.1007/s00216-014-7790-z
Human kinases are one of the most promising targets for cancer therapy. Methods able to measure the effects of drugs on these cell agents remain crucial for biologists and medicinal chemists. The current work therefore sought to develop an in-capillary enzymatic assay based on capillary electrophoresis (CE) to evaluate the inhibition of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), and the mammalian target of rapamycin (mTOR). These kinases belong to the same signaling pathway PI3K/Akt/mTOR. For this proposal, the capillary was used as a nanoreactor in which a few nanoliters of the kinase, its substrate, adenosine triphosphate (ATP), and the potent inhibitor were separately injected. A transverse diffusion of laminar flow profiles (TDLFP) approach was employed to mix the reactants. Adenosine diphosphate (ADP ) was detected online at 254 nm. The CE assay was first developed on the alpha isoform of PI3K. It was compared to five commercial kits frequently used to assess kinase inhibition, based on time-resolved fluorescence resonance energy transfer (TR-FRET) and bioluminescence. Each assay was evaluated in terms of sensitivity (S/B), reproducibility (Z’), and variability (r (2)). This CE method was easily extended to assay the inhibition of the beta, gamma, and delta isoforms of PI3K, and of the other kinases of the pathway, Akt1 and mTOR, since it is based on in-capillary mixing by TDLFP and on ADP quantification by simple UV absorption. This work shows for the first time the evaluation of inhibitors of the kinases of the PI3K/Akt/mTOR pathway using a common in-capillary CE assay. Several inhibitors with a wide range of affinity toward these enzymes were tested.
Design, Synthesis, and Biological Activity of Pyridopyrimidine Scaffolds as Novel PI3K/mTOR Dual InhibitorsJournal of Medicinal Chemistry (2014) 57 (3) 613-631 - doi : 10.1021/jm401138v
The design, synthesis, and screening of dual PI3K/mTOR inhibitors that gave nanomolar enzymatic and cellular activities on both targets with an acceptable kinase selectivity profile are described. A docking study was performed to understand the binding mode of the compounds and to explain the differences in biological activity. In addition, cellular effects of the best dual inhibitors were determined on six cancer cell lines and compared to those on a healthy diploid cell line for cellular cytotoxicity. Two compounds are highly potent on cancer cells in the submicromolar range without any toxicity on healthy cells. A more detailed analysis of the cellular effect of these PI3K/mTOR dual inhibitors demonstrated that they induce GI-phase cell cycle arrest in breast cancer cells and trigger apoptosis. These compounds show an interesting kinase profile as dual PI3K/mTOR tool compounds or as a chemical series for further optimization to progress into in vivo experiments.
Héricourt, F., Chefdor, F., Bertheau, L., Tanigawa, M., Maeda, T., Guirimand, G., Courdavault, V., Larcher, M., Depierreux, C., Bénédetti, H., Morabito, D., Brignolas, F., Carpin, S. (2013)
Characterization of histidine-aspartate kinase HK1 and identification of histidine phosphotransfer proteins as potential partners in a Populus multistep phosphorelayPhysiologia Plantarum (2013) 149 (2) 188-199 - doi : 10.1111/ppl.12024
In poplar, we identified proteins homologous to yeast proteins involved in osmosensing multistep phosphorelay Sln1p-Ypd1p-Ssk1p. This finding led us to speculate that Populus cells could sense osmotic stress by a similar mechanism. This study focuses on first and second protagonists of this possible pathway : a histidine-aspartate kinase (HK1), putative osmosensor and histidine phosphotransfer proteins (HPt1 to 10), potential partners of this HK. Characterization of HK1 showed its ability to homodimerize in two-hybrid tests and to act as an osmosensor with a kinase activity in yeast, by functional complementation of sln1Δ sho1Δ strain. Moreover, in plant cells, plasma membrane localization of HK1 is shown. Further analysis on HPts allowed us to isolate seven new cDNAs, leading to a total of 10 different HPts identified in poplar. Interaction tests showed that almost all HPts can interact with HK1, but two of them exhibit stronger interactions, suggesting a preferential partnership in poplar. The importance of the phosphorylation status in these interactions has been investigated with two-hybrid tests carried out with mutated HK1 forms. Finally, in planta co-expression analysis of genes encoding these potential partners revealed that only three HPts are co-expressed with HK1 in different poplar organs. This result reinforces the hypothesis of a partnership between HK1 and these three preferential HPts in planta. Taken together, these results shed some light on proteins partnerships that could be involved in the osmosensing pathway in Populus.
Opposing Mcl-1, the GALIG proapoptotic gene is upregulated as neutrophils die and underexpressed in Acute Myeloid Leukemia cellsMolecular Immunology 56 (1-2) 123-128
GALIG gene expression induces apoptosis in cultured cells through a pathway still under investigation. It is highly expressed in leukocytes but weakly detectable in bone marrow, suggesting a role in the myeloid lineage homeostasis. We show here that GALIG-induced cell death is counteracted by the overexpression of MCL-1, a pro-survival member of the Bcl2 family. Moreover, during spontaneous neutrophil apoptosis, a substantial increase in GALIG gene expression is observed : GALIG still opposes MCL-1. Finally, in bone marrow and peripheral blood cells from patients with Acute Myeloid Leukemia type 2, the level of GALIG transcripts is massively down-regulated when compared to their normal counterparts, while MCL-1 is expressed to the same extent. These data suggest that GALIG could be a key player in the cell death pathway involved in leukocytes homeostasis and myeloid malignancies.
Mitogaligin is a mitochondrion-targeting protein involved in cell death. The sequence of the protein is unrelated to that of any known pro- or antiapoptotic protein. Mitochondrial targeting is controlled by an internal sequence from residues 31 to 53, and although this sequence is essential and sufficient to provoke cell death, the precise mechanism of action at the mitochondrial membrane remains to be elucidated. Here, by focusing on the [31–53] fragment, we first assessed and confirmed its cell cytotoxicity by microinjection. Subsequently, with the aid of membrane models, we evaluated the impact of the membrane environment on the 3D structure of the peptide and on how the peptide is embedded and oriented within membranes. The fragment is well organized, even though it does not contain a canonical secondary structure, and adopts an interfacial location. Structural comparison with other membrane-interacting Trp-rich peptides demonstrated similarities with the antimicrobial peptide tritrpcidin.
Bruel A., Logé C., Tauzia M.-L., de Ravache M., Le Guevel R., Guillouzo C., Lohier J.-F., de Oliveira Santos J. S., Lozach O., Meijer L., Ruchaud S., Bénédetti H. and Robert J.-M. (2012)
Synthesis and biological evaluation of new 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles as PI3Kα inhibitorsEuropean Journal of Medicinal Chemistry (2012) 57, 225-233 - doi :
A series of novel 5-benzylated 4-oxo-3,4-dihydro-5H-pyridazino[4,5-b]indoles was synthesized through a newly developed approach. All these compounds were evaluated against DYRK1A, CDK5 and PI3Kα and showed promising inhibitory activities against PI3Kα with most IC50 values in the micromolar range. Among them, compound 18 was strongly considered as the most interesting compound with an IC50 value of 0.091 μM. This series exhibited also significant anti-proliferative effects in various human cancer cell lines including those resulting in activation of the PI3K pathway.
Ras GTPases mediate numerous biological processes through their ability to cycle between an inactive GDP-bound form and an active GTP-bound form. Guanine nucleotide exchange factors (GEFs) favor the formation of the active Ras-GTP, whereas GTPase activating proteins (GAPs) promote the formation of inactive Ras-GDP. Numerous studies have established complex signaling cross-talks between Ras GTPases and other members of the superfamily of small GTPases. GEFs were thought to play a major role in these cross-talks. However, recently GAPs were also shown to play crucial roles in these processes. Among RasGAPs, Nf1 is of special interest. Nf1 is responsible for the genetic disease Neurofibromatosis type I, and recent data strongly suggest that this RasGAP connects different signaling pathways.
METHODOLOGY/PRINCIPAL FINDINGS :
In order to know if the RasGAP Nf1 might play a role in connecting Ras GTPases to other small GTPase pathways, we systematically looked for new partners of Nf1, by performing a yeast two-hybrid screening on its SecPH domain. LIMK2, a major kinase of the Rho/ROCK/LIMK2/cofilin pathway, was identified in this screening. We confirmed this interaction by co-immunoprecipitation experiments, and further characterized it. We also demonstrated its specificity : the close related homolog of LIMK2, LIMK1, does not interact with the SecPH domain of Nf1. We then showed that SecPH partially inhibits the kinase activity of LIMK2 on cofilin. Our results furthermore suggest a precise mechanism for this inhibition : in fact, SecPH would specifically prevent LIMK2 activation by ROCK, its upstream regulator.
Although previous data had already connected Nf1 to actin cytoskeleton dynamics, our study provides for the first time possible detailed molecular requirements of this involvement. Nf1/LIMK2 interaction and inhibition allows to directly connect neurofibromatosis type I to actin cytoskeleton remodeling, and provides evidence that the RasGAP Nf1 mediates a new cross-talk between Ras and Rho signaling pathways within the superfamily of small GTPases.
LIMK2d, a truncated isoform of Lim kinase 2 regulates neurite growth in absence of the LIM kinase domainBiochemical and Biophysical Research Communications 420 (2) 247–252
Lim kinase 2 isoforms, LIMK2a and LIMK2b, phosphorylate cofilin leading to remodeling of actin cytoskeleton during neuronal differentiation. The expression and function of the LIMK2d isoform, missing the kinase domain, remain unknown. We analyzed the expression of LIMK2 splice variants in adult rat brain and in cultures of rat neural stem cells by RT-QPCR. All three splice variants were expressed in adult cortex, hippocampus and cerebellum. Limk2a and Limk2d expression, but not Limk2b, increased during neuronal differentiation. We studied the localization and function of LIMK2d isoform by transfecting Hela, NSC-34, and hippocampal rat neuron cultures. Similarly to LIMK2b, LIMK2d was expressed in the cytoplasm, neurites and dendritic spines, but not in the nucleus. Similarly to LIMK2a, LIMK2d over-expression in NSC-34 cells increased neurite length, but independently of cofilin phosphorylation or of direct interaction with actin. Overall, these results indicate that LIMK2d is a third LIMK2 isoform which regulates neurite extension and highlights the possible existence of a kinase independent function of LIMK2.
Plant LIM domain proteins may act as transcriptional activators of lignin biosynthesis and/or as actin binding and bundling proteins. Plant LIM genes have evolved in phylogenetic subgroups differing in their expression profiles : in the whole plant or specifically in pollen. However, several poplar PtLIM genes belong to uncharacterized monophyletic subgroups and the expression patterns of the LIM gene family in a woody plant have not been studied.
In this work, the expression pattern of the twelve duplicated poplar PtLIM genes has been investigated by semi quantitative RT-PCR in different vegetative and reproductive tissues. As in other plant species, poplar PtLIM genes were widely expressed in the tree or in particular tissues. Especially, PtXLIM1a, PtXLIM1b and PtWLIM1b genes were preferentially expressed in the secondary xylem, suggesting a specific function in wood formation. Moreover, the expression of these genes and of the PtPLIM2a gene was increased in tension wood. Western-blot analysis confirmed the preferential expression of PtXLIM1a protein during xylem differentiation and tension wood formation. Genes classified within the pollen specific PLIM2 and PLIM2-like subgroups were all strongly expressed in pollen but also in cottony hairs. Interestingly, pairs of duplicated PtLIM genes exhibited different expression patterns indicating subfunctionalisations in specific tissues.
The strong expression of several LIM genes in cottony hairs and germinating pollen, as well as in xylem fibers suggests an involvement of plant LIM domain proteins in the control of cell expansion. Comparisons of expression profiles of poplar LIM genes with the published functions of closely related plant LIM genes suggest conserved functions in the areas of lignin biosynthesis, pollen tube growth and mechanical stress response. Based on these results, we propose a novel nomenclature of poplar LIM domain proteins.
The PEBP (phosphatidylethanolamine-binding protein) family is a large group of proteins whose human member, hPEBP1, has been shown to play multiple functions, influencing intracellular signaling cascades, cell cycle regulation, neurodegenerative processes, and reproduction. It also acts, by an unknown mechanism, as a metastasis suppressor in a number of cancers. A more complete understanding of its biological role is thus necessary. As the yeast Saccharomyces cerevisiae is a powerful and easy to handle model organism, we focused on Tfs1p, the yeast ortholog of hPEBP1. In a previous study based on a two-hybrid approach, we showed that Tfs1p interacts and inhibits Ira2p, a GTPase Activating Protein (GAP) of the small GTPase Ras. In order to further characterize the molecular functions of Tfs1p, we undertook the identification of protein complexes formed around Tfs1p using a targeted proteomics approach. Complexed proteins were purified by tandem-affinity, cleaved with trypsin, and identified by nanoflow liquid chromatography coupled with tandem mass spectrometry. Overall, 14 new interactors were identified, including several proteins involved in intermediate metabolism. We confirmed by co-immunoprecipitation that Tfs1p interacts with Glo3p, a GAP for Arf GTPases belonging to the Ras superfamily of small GTPases, indicating that Tfs1p may be involved in the regulation of another GAP. We similarly confirmed the binding of Tfs1p with the metabolic enzymes Idp1p and Pro1p. Integration of these results with known functional partners of Tfs1p shows that two subnetworks meet through the Tfs1p node, suggesting that it may act as a bridge between cell signaling and intermediate metabolism in yeast.
A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell lineBiochemical and Biophysical Research Communications 418 (4) 689-694
Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line : CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using ‘‘in situ’’ proximity ligation assay (PLA).
Robinet, P., Mollet, L., Gonzalez, P., Normand, T., Charpentier, S., Brule, F., Dubois, M. & Legrand, A. (2010)
Gombault, A., Warringer, J., Caesar, R., Godin, F., Vallee, B., Doudeau, M., Chautard, H., Blomberg, A. & Benedetti, H. (2009)
Multiple reprobing of Western blots after inactivation of peroxidase activity by its substrate, hydrogen peroxide.Anal. Biochem. 393, 129-131. Gonzalez, P., Robinet, P., Charpentier, S., Mollet, L., Normand, T., Dubois, M. & Legrand, A. (2009)
Davenas E, Rouleau A, Morisset S, and Arrang JM. (2008)
Gombault, A ; Godin, F ; Sy, D ; Legrand, B ; Chautard, H ; Vallee, B ; Vovelle, F ; Benedetti, H (2007)
Molecular basis of the Tfs1/Ira2 interaction : A combined protein engineering and molecular modelling studyJournal of Molecular Biology 374 (3) 604-617
Tfs1p and Ylr179cp are yeast proteins belonging to the PEBP family. Tfs1p, but not Ylr179cp, has been shown to interact with and inhibit Ira2p, a GTPase-activating protein of Ras. Tfs1p has been shown to be a specific inhibitor of the CPY protease and the 3D structure of the complex has been resolved. To shed light on the molecular determinants of Tfs1p involved in the Tfs1/Ira2 interaction, the 3D structure of Ylr179cp has been modelled and compared to that of Tfs1p. Tfs1p point mutants and Tfs1 hybrid proteins combining regions of Tfs1p and Ylr179cp were also designed and their function was tested. Results, interpreted from a structural point of view, show that the accessibility of the surface pocket of Tfs1p, its N-terminal region and the specific electrostatic properties of a large surface region containing these two elements, play a crucial role in this interaction. (c) 2007 Elsevier Ltd. All rights reserved.
Destabilization of membranes containing cardiolipin or its precursors by peptides derived from mitogaligin, a cell death proteinBiochemistry 46 (25) 7374-7382
Galig, a gene embedded within the galectin-3 gene, induces cell death when transfected in human cells. This death is associated with cell shrinkage, nuclei condensation, and aggregation of mitochondria. Galig contains two different overlapping open reading frames encoding two unrelated proteins. Previous observations have shown that one of these proteins, named mitogaligin, binds to mitochondria and promotes the release of cytochrome c. However, the mechanism of action of this cytotoxic protein remains still obscure.
Histamine H3 and dopamine D2 receptor-mediated [35S]GTPg[S]binding in rat striatum : evidence for additive effects but lack of interactionsBiochem Pharmacol (2007), 73 (8) : 1172-1181 Arrang J, Morisset S and Gbahou F. (2007)
Chefdor, F ; Benedetti, H ; Depierreux, C ; Delmotte, F ; Morabito, D ; Carpin, S (2006)
To study the Populits response to an osmotic stress, we have isolated one cDNA encoding a histidine-aspartate kinase (HK1) and four cDNAs encoding histidine-containing phosphotransfer proteins (HPts), HPt1-4. The predicted HK1 protein shares a typical structure with ATHK1 and SLN1 osmosensors. The 4 HPTs are characterized by the histidine phosphotransfer domain. We have shown that HK1 is upregulated during an osmotic stress in hydroponic culture. We have detected an interaction between HK1 and HPt2, using the yeast two-hybrid system. These results suggest the existence of a multi-step phosphorelay pathway probably involved in osmotic stress sensing in Populus. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Vallee, B ; Riezman, H (2005)
Ceramide plays a crucial role as a basic building block of sphingolipids, but also as a signalling molecule mediating the fate of the cell. Although Lac1p and Lag1p have been shown recently to be involved in acyl- CoA- dependent ceramide synthesis, ceramide synthase is still poorly characterized. In this study, we expressed tagged versions of Lac1p and Lag1p and purified them to near homogeneity.
Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector.
Yhr049w/FSH1 was recently identified in a combined computational and experimental proteomics analysis for the detection of active serine hydrolases in yeast. This analysis suggested that FSH1 might be a serine-type hydrolase belonging to the broad functional alpha beta-hydrolase superfamily. In order to get insight into the molecular function of this gene, it was targeted in our yeast structural genomics project. The crystal structure of the protein confirms that it contains a Ser/His/Asp catalytic triad that is part of a minimal alpha beta-hydrolase fold. The architecture of the putative active site and analogies with other protein structures suggest that FSH1 may be an esterase. This finding was further strengthened by the unexpected presence of a compound covalently bound to the catalytic serine in the active site. Apparently, the enzyme was trapped with a reactive compound during the purification process.
Chautard, H ; Jacquet, M ; Schoentgen, F ; Bureaud, N ; Benedetti, H (2004)
Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p Ras GTPase-activating protein in Saccharomyces cerevisiaeEukaryotic Cell 3 (2) 459-470
Ras proteins are guanine nucleotide-binding proteins that are highly conserved among eukaryotes. They are involved in signal transduction pathways and are tightly regulated by two sets of antagonistic proteins : GTPase-activating proteins (GAPs) inhibit Ras proteins, whereas guanine exchange factors activate them. In this work, we describe Tfs1p, the first physiological inhibitor of a Ras GAP, Ira2p, in Saccharomyces cerevisiae. TFS1 is a multicopy suppressor of the cdc25-1 mutation in yeast and corresponds to the so-called Ic CPY cytoplasmic inhibitor.
Train A, an RNase a-like protein without RNase activity, is secreted and reabsorbed by the same epididymal cells under testicular controlBiology of Reproduction 71 (5) 1677-1687
Most of the proteins secreted in the epididymis are produced by the proximal region, and several of them are secreted in abundance. Many of these major proteins have now been identified, including a new epididymis-specific RNase A-like Train A protein, which has been recently described in several mammals. This protein is expressed and secreted exclusively in the initial part of the epididymis. RNase A activity was analyzed in the fluids from the testis and from different epididymal regions, but in no case was the Train A protein found to have RNase A activity. The protein was present only in the luminal fluid of the epididymal region that secreted it. Using an in vitro/in vivo microperfusion technique and immunogold electron microscopy labeling, we demonstrated that the epithelium that secreted it specifically reabsorbed the protein that was present in the lumen of the tubule.
Funato, K., Lombardi, R., Vallée, B. & Riezman, H. (2003)
Lcb4p is a Key Regulator of Ceramide Synthesis from Exogenous Long Chain Sphingoid Base in Saccharomyces cerevisiae.J.Biol. Chem. 278, 7325-7334. Carlier, M., Cohen-Salmon, C., Cherif, C., Verray, F.M., Arechi, P., Godin, F., Sluyter, F., Marcet, B., Verrier, B. & Roubertoux, P.L. (2003)
Development of congenic strains for mitochondrial DNA in mice : implication of mtDNA in pre-wearing development and motor behaviour.Behavior Gentics 33, 699-700 Roubertoux, P.L., Sluyter, F., Carlier, M., Marcet, B., Maaroud-Veray, F., Cherif, C., Marican, C., Arrechi, P., Godin, F., Jamon, M., Verrier, B. & Cohen-Salmon, C. (2003)
Peptides corresponding to the N- and C-terminal parts of PEBP are well-structured in solution : new insights into their possible interaction with partners in vivoJournal of Peptide Research 61 (2) 47-57
Recently, it has been shown that mammalian PEBPs are implicated in several signalling pathways controlling the cellular cycle. In particular, during brain development, the N-terminal part of mammalian PEBP is specifically cleaved and the resulting 11 amino acid peptide stimulates the growth and activity of acetylcholinergic neurons. The crystallographic structure of bovine and human PEBPs has revealed that their N- and C-terminal parts are accessible and exposed to the solvent suggesting that they may be involved in specific interactions with cellular partners. We have chemically synthetized the two peptides corresponding to these terminal parts and studied their structure in solution by circular dichroism and NMR spectroscopies : both of them are well-structured.
Tfs1p, a member of the PEBP family, inhibits the Ira2p but not the Ira1p Ras GTPase activating protein in Saccharomyces cerevisiae.Yeast 20 S161-S161 Suppl. 1 Schwartz JC, Morisset S, Rouleau A, Ligneau X, Gbahou F, Tardivel-Lacombe J, Stark H, Schunack W, Ganellin CR and Arrang JM. (2003)
Therapeutic implications of constitutive activity of receptors : the example of the histamine H3 receptorJ Neural Transm (2003) Suppl:1-16 Ilien B., Franchet C., Bernard P., Morisset S., Weill C. Bourguignon J., Hibert M. and Galzi J.L. (2003)
Fluorescence resonance energy transfer to probe human muscarinic M1 receptor structure and drug binding propertiesJ. Neurochem. (2003) 85:768-778 Gbahou F, Rouleau A, Morisset S, Parmentier R, Crochet S, Lin JS, Ligneau X, Tardivel-Lacombe J, Stark H, Schunack W, Ganellin CR, Schwartz JC and Arrang JM. (2003)
Funato, K ; Vallee, B ; Riezman, H (2002)
We compared deleted copies of the seven mauritiana subfamilies of mariner transposable elements in species of the Drosophilidae. All elements were detected by PCR using the inverted terminal repeats of the Mos1 element of Drosophila mauritiana as primers. A higher frequency of breakpoints in the 5’ part of the element compared to the 3’ part was observed. Of the 27 deletions, 9 (33%) occurred between short direct repeats (SDR) of 5 to 8 bp. The SDRs can be at or close to the breakpoints of the deletion. A deleted copy of D. simulans (St. Martin population) had three repeats of a motif present only once in the complete consensus sequence. The high frequency of SDRs at or near the breakpoints of the deletions strongly suggests that some of them do not occur at random. Mechanisms that might explain these deletions, such as unequal crossing-over, ectopic recombination, and abortive gap repair, are discussed.
Analysis of the Escherichia coli Tol-Pal and TonB systems by periplasmic production of Tol, TonB, colicin, or phage capsid soluble domainsBiochimie 84 (5-6) 413-421
The aim of this review is to describe an in vivo assay of the interactions taking place in the Tol-Pal or TonB-ExbB-ExbD envelope complexes in the periplasm of Escherichia coli and between them and colicins or g3p protein of filamentous bacteriophages. Domains of colicins or periplasmic soluble domains of Tol or TonB proteins can be artificially addressed to the periplasm of bacteria by fusing them to a signal sequence from an exported protein. These domains interact specifically in the periplasm with the Tol or TonB complexes and disturb their function, which can be directly detected by the appearance of specific tol or tonB phenotypes. This technique can be used to detect new interactions, to characterize them biochemically and to map them or to induce tol or tonB phenotypes to study the functions of these two complexes. 0 2002 Societe francaise de biochimie et biologie moleculaire / Editions scientifiques et medicales Elsevier SAS. All rights reserved.
Histamine H3-receptor-mediated [35S]GTP gamma[S] binding : evidence for constitutive activity of the recombinant and native rat and human H3 receptorsBr J Pharmacol. (2002) 135, 383-392 Morisset S., Pilon C., Tardivel-Lacombe J., Weinstein D., Rostene W., Betancur C., Sokoloff P., Schwartz J.C. and Arrang J.M. (2002)
Acute and chronic effects of methamphetamine on tele-methylhistamine levels in mouse brain : selective involvement of the D(2) and not D(3) receptorJ. Pharmacol. Exp. Ther. (2002) 300, 621-628 Chotard C., Ouimet T., Morisset S., Sahm U., Schwartz J.C. and Tottier S. (2002)
Effects of histamine H3 receptor agonist and antagonist on histamine co-transmitter expression in rat brainJ. Neural. Transm. (2002) 109, 293-306
Vallee, B ; Schorling, S ; Barz, WP ; Riezman, H ; Oesterhelt, D (2001)
Lag1p and Lac1p are essential for the acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisaeMolecular Biology of The Cell 12 (11) 3417-3427
Lag1p and Lac1p are two homologous transmembrane proteins of the endoplasmic reticulum in Saccharomyces cerevisiae. Homologous genes have been found in a wide variety of eukaryotes. In yeast, both genes, LAC1 and LAG1, are required for efficient endoplasmic reticulum-to-Golgi transport of glycosylphosphatidylinositol-anchored proteins. In this study, we show that lag1 Delta lac1 Delta cells have reduced sphingolipid levels due to a block of the fumonisin B1-sensitive and acyl-CoA-dependent ceramide synthase reaction.
Behaviour of bovine phosphatidylethanolamine-binding protein with model membranes - Evidence of affinity for negatively charged membranesEuropean Journal of Biochemistry 268 (22) 5831-5841
The ability of phosphatidylethanolamine-binding protein (PEBP) to bind membranes was tested by using small and large unilamellar vesicles and monolayers composed Of L-a -1,2-dimyristoylphosphatidylcholine, L-a -1,2-dimyristoylphosphatidylglycerol and L-a -1,2-dimyristoylphosphatidylethanolamine. PEBP only bound to model membranes containing L-a -1,2-dimyristoylphosphatidylglycerol ; the interaction was primarily due to electrostatic forces between the basic protein and the acidic phospholipids. Further experiments indicated that the interaction was not dependent on the length and unsaturation of the phospholipid acyl chains and was not modified by the presence of cholesterol in the membrane.
Identification of an internal gene to the human galectin-3 gene with two different overlapping reading frames that do not encode galectin-3Journal of Biological Chemistry 276 (4) 2652-2657
We previously reported that alternative transcripts were initiated within the second intron of the human Galectin-3 gene (LGALS3), We now demonstrate that these transcripts arise from an internal gene embedded within LGALS3 and named galig (Galectin-3 internal gene). Tissue-specific expression of galig was assayed by screening of several human tissues. Contrary to LGALS3, galig appears to be tightly regulated and principally activated in leukocytes from peripheral blood. Cloning and characterization of galig transcripts revealed that they contain two out-of-frame overlapping open-reading frames (ORFs). Transfection of expression vectors encoding enhanced green fluorescent protein (EGFP) chimeras indicated that both ORFs could be translated in proteins unrelated to Galectin-3. The ORF1 polypeptide targets EGFP to cytosol and nucleus whereas ORF2 targets EGFP to mitochondria. These results revealed the exceptional genetic organization of the LGALS3 locus.
Crystal structures of YBHB and YBCL from Escherichia coli, two bacterial homologues to a Raf kinase inhibitor proteinJournal of Molecular Biology 310 (3) 617-634
In rat and human cells, RKIP (previously known as PEEP) was characterized as an inhibitor of the MEK phosphorylation by Raf-l. In Escherichia coli, the genes ybhb and ybcl possibly encode two RKIP homologues while in the genomes of other bacteria and archaebacteria other homologous genes of RKIP have been found. The parallel between the cellular signaling mechanisms in eukaryotes and prokaryotes suggests that these bacterial proteins could be involved in the regulation of protein phosphorylation by kinases as well. We first showed that the proteins YBHB and YBCL were present in the cytoplasm and periplasm of E. coli, respectively, after which we determined their crystallographic structures.
Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasmMolecular Microbiology 42 (2) 331-344
Several proteins of the Tol/Pal system are required for group A colicin import into Escherichia coli. Colicin A interacts with TolA and TolB via distinct regions of its N-terminal domain. Both interactions are required for colicin translocation. Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR. This interaction is specific to colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the colicin translocation step. TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm. The interaction involves part of the periplasmic domain of TolR and a small region of the colicin A N-terminal domain. This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the colicin A N-terminal domain and appear to be arranged linearly in the colicin sequence. Multiple contacts with periplasmic-exposed Tol proteins are therefore a general principle required for group A colicin translocation.
The histamine H3 receptor : gene organization, multiple isoforms, constitutive activity and molecular pharmacologyDans ‘’Histamine Research in the New Millenium’’. Edité par T. Watanabe, H. Timmermann et K. Yanai. Elsevier, Amsterdam, (2001), 9-21
Lazdunski, C ; Bouveret, E ; Rigal, A ; Journet, L ; Lloubes, R ; Benedetti, H (2000)