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Pierre Legrain

Publications and source records attributed to Pierre Legrain.

16 recordsLinked to original sources
PubMed2005

ARHGAP10 is necessary for alpha-catenin recruitment at adherens junctions and for Listeria invasion.

E-cadherin mediates the formation of adherens junctions between epithelial cells. It serves as a receptor for Listeria monocytogenes, a bacterial pathogen that enters epithelial cells. The L. monocytogenes surface protein, InlA, interacts with the extracellular domain of E-cadherin. In adherens junctions, this ectodomain is involved in homophilic interactions whereas the cytoplasmic domain binds beta-catenin, which then recruits alpha-catenin. alpha-catenin binds to actin directly, or indirectly, thus linking E-cadherin to the actin cytoskeleton. Entry of L. monocytogenes into cells and adherens junction formation are dynamic events that involve actin and membrane rearrangements. To understand these processes better, we searched for new ligands of alpha-catenin. Using a two-hybrid screen, we identified a new partner of alpha-catenin: ARHGAP10. This protein colocalized with alpha-catenin at cell-cell junctions and was recruited at L. monocytogenes entry sites. In ARHGAP10-knockdown cells, L. monocytogenes entry and alpha-catenin recruitment at cell-cell contacts were impaired. The GAP domain of ARHGAP10 has GAP activity for RhoA and Cdc42. Its overexpression disrupted actin cables, enhanced alpha-catenin and cortical actin levels at cell-cell junctions and inhibited L. monocytogenes entry. Altogether, our results show that ARHGAP10 is a new component of cell-cell junctions that controls alpha-catenin recruitment and has a key role during L. monocytogenes uptake.

Actins↗
PubMed2005

The Shigella flexneri effector OspG interferes with innate immune responses by targeting ubiquitin-conjugating enzymes.

Bacteria of Shigella spp. are responsible for shigellosis in humans. They use a type III secretion system to inject effector proteins into host cells and induce their entry into epithelial cells or trigger apoptosis in macrophages. We present evidence that the effector OspG is a protein kinase that binds various ubiquitinylated ubiquitin-conjugating enzymes, including UbcH5, which belongs to the stem cell factor SCF(beta-TrCP) complex promoting ubiquitination of phosphorylated inhibitor of NF-kappaB type alpha (phospho-IkappaBalpha). Transfection experiments indicated that OspG can prevent phospho-IkappaBalpha degradation and NF-kappaB activation induced by TNF-alpha stimulation. Infection of epithelial cells by the S. flexneri wild-type strain, but not an ospG mutant, led to accumulation of phospho-IkappaBalpha, consistent with OspG inhibiting SCF(beta-TrCP) activity. Upon infection of ileal loops in rabbits, the ospG mutant induced a stronger inflammatory response than the wild-type strain. This finding indicates that OspG negatively controls the host innate response induced by S. flexneri upon invasion of the epithelium.

Animals↗
PubMed2005

PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa.

By using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.

Animals↗
PubMed2005

Protein interaction mapping: a Drosophila case study.

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.

Animals↗
PubMed2004

Myosin XVa and whirlin, two deafness gene products required for hair bundle growth, are located at the stereocilia tips and interact directly.

Defects in myosin XVa and the PDZ domain-containing protein, whirlin, underlie deafness in humans and mice. Hair bundles of mutant mice defective for either protein have abnormally short stereocilia. Here, we show that whirlin, like myosin XVa, is present at the very tip of each stereocilium in the developing and mature hair bundles of the cochlear and vestibular system. We found that myosin XVa SH3-MyTH4 region binds to the short isoform of whirlin (PR-PDZ3) that can rescue the stereocilia growth defect in whirlin defective mice. Moreover, the C-terminal MyTH4-FERM region of myosin XVa binds to the PDZ1 and PDZ2 domains of the long whirlin isoform. We conclude that a direct myosin XVa-whirlin interaction at the stereocilia tip is likely to control the elongation of stereocilia. Whirlin, unlike myosin XVa, is also transiently localized in the basal region of developing stereocilia in rat vestibular and cochlear hair cells until P4 and P12, respectively. Notably, whirlin also interacts with myosin VIIa that is present along the entire length of the stereocilia. Finally, we show that the transmembrane netrin-G1 ligand (NGL-1) binds to the PDZ1 and PDZ2 domains of whirlin and has an extracellular region that homophilically self-interacts in a Ca2+-dependent manner. The interaction between whirlin and NGL-1 might be involved in the stabilization of interstereociliar links.

Actins↗
PubMed2004

Biochemical characterization of protein complexes from the Helicobacter pylori protein interaction map: strategies for complex formation and evidence for novel interactions within type IV secretion systems.

We have investigated a large set of interactions from the Helicobacter pylori protein interaction map previously identified by high-throughput yeast two-hybrid (htY2H)-based methods. This study had two aims: i) to validate htY2H as a source of protein-protein interaction complexes for high-throughput biochemical and structural studies of the H. pylori interactome; and ii) to validate biochemically interactions shown by htY2H to involve components of the H. pylori type IV secretion systems. Thus, 17 interactions involving 31 proteins and protein fragments were studied, and a general strategy was designed to produce protein-interacting partners for biochemical and structural characterization. We show that htY2H is a valid source of protein-protein complexes for high-throughput proteome-scale characterization of the H. pylori interactome, because 76% of the interactions tested were confirmed biochemically. Of the interactions involving type IV secretion proteins, three could be confirmed. One interaction is between two components of the type IV secretion apparatus, ComB10 and ComB4, which are VirB10 and VirB4 homologs, respectively. Another interaction is between a type IV component (HP0525, a VirB11 homolog) and a non-type IV secretion protein (HP01451), indicating that proteins other than the core VirB (1-11)-VirD4 proteins may play a role in type IV secretion. Finally, a third interaction was biochemically confirmed between CagA, a virulence factor secreted by the type IV secretion system encoded by the Cag pathogenicity island, and a non-type IV secretion protein, HP0496.

Bacterial Proteins↗
PubMed2004

The HUPO PSI's molecular interaction format--a community standard for the representation of protein interaction data.

A major goal of proteomics is the complete description of the protein interaction network underlying cell physiology. A large number of small scale and, more recently, large-scale experiments have contributed to expanding our understanding of the nature of the interaction network. However, the necessary data integration across experiments is currently hampered by the fragmentation of publicly available protein interaction data, which exists in different formats in databases, on authors' websites or sometimes only in print publications. Here, we propose a community standard data model for the representation and exchange of protein interaction data. This data model has been jointly developed by members of the Proteomics Standards Initiative (PSI), a work group of the Human Proteome Organization (HUPO), and is supported by major protein interaction data providers, in particular the Biomolecular Interaction Network Database (BIND), Cellzome (Heidelberg, Germany), the Database of Interacting Proteins (DIP), Dana Farber Cancer Institute (Boston, MA, USA), the Human Protein Reference Database (HPRD), Hybrigenics (Paris, France), the European Bioinformatics Institute's (EMBL-EBI, Hinxton, UK) IntAct, the Molecular Interactions (MINT, Rome, Italy) database, the Protein-Protein Interaction Database (PPID, Edinburgh, UK) and the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, EMBL, Heidelberg, Germany).

Database Management Systems↗
PubMed2004

Functional proteomics mapping of a human signaling pathway.

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Antiviral drug discovery strategy using combinatorial libraries of structurally constrained peptides.

We have developed a new strategy for antiviral peptide discovery by using lyssaviruses (rabies virus and rabies-related viruses) as models. Based on the mimicry of natural bioactive peptides, two genetically encoded combinatorial peptide libraries composed of intrinsically constrained peptides (coactamers) were designed. Proteomic knowledge concerning the functional network of interactions in the lyssavirus transcription-replication complex highlights the phosphoprotein (P) as a prime target for inhibitors of viral replication. We present an integrated, sequential drug discovery process for selection of peptides with antiviral activity directed against the P. Our approach combines (i). an exhaustive two-hybrid selection of peptides binding two phylogenetically divergent lyssavirus P's, (ii). a functional analysis of protein interaction inhibition in a viral reverse genetic assay, coupled with a physical analysis of viral nucleoprotein-P complex by protein chip mass spectrometry, and (iii). an assay for inhibition of lyssavirus infection in mammalian cells. The validity of this strategy was demonstrated by the identification of four peptides exhibiting an efficient antiviral activity. Our work highlights the importance of P as a target in anti-rabies virus drug discovery. Furthermore, the screening strategy and the coactamer libraries presented in this report could be considered, respectively, a general target validation strategy and a potential source of biologically active peptides which could also help to design pharmacologically active peptide-mimicking molecules. The strategy described here is easily applicable to other pathogens.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of a novel RanGTP-binding protein in the yeast Saccharomyces cerevisiae.

The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1,2 ) and nuclear envelope formation (3,4 ) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle.

Active Transport, Cell Nucleus↗
PubMed2002

Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle.

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.

Actins↗
PubMed2002

Comprehensive proteomic analysis of breast cancer cell membranes reveals unique proteins with potential roles in clinical cancer.

Proteins associated with cancer cell plasma membranes are rich in known drug and antibody targets as well as other proteins known to play key roles in the abnormal signal transduction processes required for carcinogenesis. We describe here a proteomics process that comprehensively annotates the protein content of breast tumor cell membranes and defines the clinical relevance of such proteins. Tumor-derived cell lines were used to ensure an enrichment for cancer cell-specific plasma membrane proteins because it is difficult to purify cancer cells and then obtain good membrane preparations from clinical material. Multiple cell lines with different molecular pathologies were used to represent the clinical heterogeneity of breast cancer. Peptide tandem mass spectra were searched against a comprehensive data base containing known and conceptual proteins derived from many public data bases including the draft human genome sequences. This plasma membrane-enriched proteome analysis created a data base of more than 500 breast cancer cell line proteins, 27% of which were of unknown function. The value of our approach is demonstrated by further detailed analyses of three previously uncharacterized proteins whose clinical relevance has been defined by their unique cancer expression profiles and the identification of protein-binding partners that elucidate potential functionality in cancer.

Amino Acid Sequence↗
PubMed2002

Prediction, assessment and validation of protein interaction maps in bacteria.

High-throughput proteomics technologies, especially the yeast two-hybrid system, produce large volumes of protein-protein interaction data organized in networks. The complete sequencing of many genomes raises questions about the extent to which such networks can be transferred between organisms. We attempted to answer this question using the experimentally derived Helicobacter pylori interaction map and the recently described interacting domain profile pair (IDPP) method to predict a virtual map for Escherichia coli. The extensive literature concerning E.coli was used to assess all predicted interactions and to validate the IDPP method, which clusters protein domains by sequence and connectivity similarities. The IDPP method has a much better heuristic value than methods solely based on protein homology. The IDPP method was further applied to Campylobacter jejuni to generate a virtual interaction map. An in-depth comparison of the chemotaxis pathways predicted in E.coli and C.jejuni led to the proposition of new functional assignments. Finally, the prediction of protein-protein interaction maps across organisms enabled us to validate some of the interactions on the original experimental map.

Bacterial Proteins↗
PubMed2002

The Shq1p.Naf1p complex is required for box H/ACA small nucleolar ribonucleoprotein particle biogenesis.

Small nucleolar ribonucleoprotein particles (snoRNPs) are essential cofactors in ribosomal RNA metabolism. Although snoRNP composition has been thoroughly characterized, the biogenesis process of these particles is poorly understood. We have identified two proteins from the yeast Saccharomyces cerevisiae, Yil104c/Shq1p and Ynl124w/Naf1p, which are essential and required for the stability of box H/ACA snoRNPs. Depletion of either Shq1p or Naf1p leads to a dramatic and specific decrease in box H/ACA snoRNA levels in vivo. A severe concomitant defect in ribosomal RNA processing is observed, consistent with the depletion of this family of snoRNAs. Shq1p and Naf1p show nuclear localization and interact with Nhp2p and Cbf5p, two core proteins of mature box H/ACA snoRNPs. Shq1p and Naf1p form a complex, but they are not strongly associated with box H/ACA snoRNPs. We propose that Shq1p and Naf1p are involved in the early biogenesis steps of box H/ACA snoRNP assembly.

Endoribonucleases↗