Osmotic stress sensing in Populus: Components identification of a phosphorelay system

AbstractTo study the Populus 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

Synthesis of new pyridazino[4,5-b]indol-4-ones and pyridazin-3(2H)-one analogs as DYRK1A inhibitors

International audienceNew 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

Insertion of Proteins into Membranes A Survey

International audienceIntegral membrane proteins are defined as proteins that span the membrane at least once. Until now, hundreds of coding sequences have been obtained for integral membrane proteins, but by contrast only a limited amount of information about the atomic structure of detergent solubilized proteins has been reported. So far, four kinds of structures have been observed for integral membrane proteins whose structures have been determined either by X-ray crystallography or electron crystallography. The structures known with high resolution are the photo-synthetic reaction centers, the porins, bacteriorhodopsin, and the light harvesting complex II. Determination of these three-dimensional (3-D) structures has provided the information upon which the extensively used prediction methods for the arrangement of membrane proteins have been based. In the absence of three-dimensional structure information, computational methods based on the analysis and comparison of amino-acid sequences have been used to predict the topology of membrane proteins. These methods give a two-dimensional picture of the arrangement of the protein in the membrane. In the meantime, new experimental procedures have been developed, increasing the possibilities to probe membrane topology, and thus the validity of the computational methods

LIM Kinases: From Molecular to Pathological Features

LIM kinases (LIMKs), LIMK1 and LIMK2, are atypical kinases, as they are the only two members of the LIM kinase family harbouring two LIM domains at their N-terminus and a kinase domain at their C-terminus [...

Insertion of Proteins into Membranes A Survey

International audienceIntegral membrane proteins are defined as proteins that span the membrane at least once. Until now, hundreds of coding sequences have been obtained for integral membrane proteins, but by contrast only a limited amount of information about the atomic structure of detergent solubilized proteins has been reported. So far, four kinds of structures have been observed for integral membrane proteins whose structures have been determined either by X-ray crystallography or electron crystallography. The structures known with high resolution are the photo-synthetic reaction centers, the porins, bacteriorhodopsin, and the light harvesting complex II. Determination of these three-dimensional (3-D) structures has provided the information upon which the extensively used prediction methods for the arrangement of membrane proteins have been based. In the absence of three-dimensional structure information, computational methods based on the analysis and comparison of amino-acid sequences have been used to predict the topology of membrane proteins. These methods give a two-dimensional picture of the arrangement of the protein in the membrane. In the meantime, new experimental procedures have been developed, increasing the possibilities to probe membrane topology, and thus the validity of the computational methods

Outils pour l'identification de ligands de LINGO-1, LINGO-2, LINGO-3 et LINGO-4, et utilisations

The present invention relates to a system comprising coupling products formed by a monomer of a protein chosen from Lingo-1, Lingo-2, Lingo-3 and Lingo-4 and by a probe emitting a signal when said monomer undergoes conformational changes, and to a screening method using said system, enabling ligands of a protein chosen from Lingo-1, Lingo-2, Lingo-3 and Lingo-4 to be identified. The present invention is industrially applicable in the field of methods for detecting molecules, for detecting interaction between molecules and for molecular screening, and also in the medical field.La présente invention se rapporte à un système comprenant des produits de couplage formés d'un monomère d'une protéine choisie parmi Lingo-1, Lingo- 2, Lingo-3 et Lingo-4 et d'une sonde émettant un signal lors de changements conformationnels dudit monomère, et un procédé de criblage utilisant ledit système et permettant d'identifier des ligands d'une protéine choisie parmi Lingo-1, Lingo-2, Lingo-3 et Lingo-4. La présente invention trouve ses applications industrielles dans le domaine des procédés de détection de molécules, de détection d'interaction entre molécules et de criblage moléculaire, ainsi que dans le domaine médical

Séminaire invité : Signalisation cellulaire et Neurofibromatose

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The Tol/PAL and TonB systems : two envelope-spanning protein complexes involved in colicin import in E. coli.

International audienceMutants in tolA, B, Q, and R genes have been isolated on the basis of their tolerance to bacterial toxins (colicins) and their resistance to the infection of filamentous phages (Ml3, fd, and fl) (Davies and Reeves, 1975a, 1975b ; Nagel de Zwaig and Luria, 1967). These genes form a cluster at 16,8 min on the chromosomal map of E. coli. tol mutants are hypersensitive to detergents and to certain drugs, and they release periplasmic proteins into the growth medium (Nagel de Zwaig and Luria, 1967). Mutations in a contiguous gene, pal, which encodes the outer membrane Peptidoglycan Associated Lipoprotein (PAL), generate a similar phenotype (Fognini-Lefebvre et al., 1987). This suggests that the Tol/PAL proteins are involved in maintaining the integrity of the outer membrane of E. coli. However, the exact physiological role of the Tol/PAL system has not yet been elucidated

Tfs1p, a Member of the PEBP Family, Inhibits the Ira2p but Not the Ira1p Ras GTPase-Activating Protein in Saccharomyces cerevisiae

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. Moreover, Tfs1p belongs to the phosphatidylethanolamine-binding protein (PEBP) family, one member of which is RKIP, a kinase and serine protease inhibitor and a metastasis inhibitor in prostate cancer. In this work, the results of (i) a two-hybrid screen of a yeast genomic library, (ii) glutathione S-transferase pulldown experiments, (iii) multicopy suppressor tests of cdc25-1 mutants, and (iv) stress resistance tests to evaluate the activation level of Ras demonstrate that Tfs1p interacts with and inhibits Ira2p. We further show that the conserved ligand-binding pocket of Tfs1—the hallmark of the PEBP family—is important for its inhibitory activity

Role of TolR N-Terminal, Central, and C-Terminal Domains in Dimerization and Interaction with TolA and TolQ

The Tol-PAL system of Escherichia coli is a multiprotein system involved in maintaining the cell envelope integrity and is necessary for the import of some colicins and phage DNA into the bacterium. It is organized into two complexes, one near the outer membrane between TolB and PAL and one in the cytoplasmic membrane between TolA, TolQ, and TolR. In the cytoplasmic membrane, all of the Tol proteins have been shown to interact with each other. Cross-linking experiments have shown that the TolA transmembrane domain interacts with TolQ and TolR. Suppressor mutant analyses have localized the TolQ-TolA interaction to the first transmembrane domain of TolQ and have shown that the third transmembrane domain of TolQ interacts with the transmembrane domain of TolR. To get insights on the composition of the cytoplasmic membrane complex and its possible contacts with the outer membrane complex, we focused our attention on TolR. Cross-linking and immunoprecipitation experiments allowed the identification of Tol proteins interacting with TolR. The interactions of TolR with TolA and TolQ were confirmed, TolR was shown to dimerize, and the resulting dimer was shown to interact with TolQ. Deletion mutants of TolR were constructed, and they allowed us to determine the TolR domains involved in each interaction. The TolR transmembrane domain was shown to be involved in the TolA-TolR and TolQ-TolR interactions, while TolR central and C-terminal domains appeared to be involved in TolR dimerization. The role of the TolR C-terminal domain in the TolA-TolR interaction and its association with the membranes was also demonstrated. Furthermore, phenotypic studies clearly showed that the three TolR domains (N terminal, central, and C terminal) and the level of TolR production are important for colicin A import and for the maintenance of cell envelope integrity