Identification of binding proteins for cholesterol absorption inhibitors as components of the intestinal cholesterol transporter

AbstractTo identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2-azetidinone cholesterol absorption inhibitors. An integral membrane protein of Mr 145.3±7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentration-dependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D-glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2-azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of Mr 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes

Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9

Symposion Forschungsdateninfrastruktur

Wie sollen qualitative sozialwissenschaftliche Daten gespeichert, archiviert und nachgenutzt werden? Das Symposion nimmt diese Frage auf und diskutiert sie anhand von Beiträgen aus Forschungsdatenzentren, die auf qualitative Daten spezialisiert sind. Neben der Vorstellung und Darstellung dieser Einrichtungen werden die mit der Archivierung einhergehenden Probleme und deren Lösungen angesprochen. How should qualitative social science data be stored, archived and re-used? The symposium takes up this question and discusses it on the basis of contributions from research data centres that specialise in qualitative data. In addition to the presentation of these institutions, the problems associated with archiving and their solutions will be discussed

Chemical synthesis of LAI-1 and amino-LAI-1.

<p>The chemical synthesis of the putative <i>L</i>. <i>pneumophila</i> small molecule compounds (A) LAI-1 and (B) amino-LAI-1 is shown.</p

Dose-dependent inhibition of chemotaxis and cell migration by LAI-1.

<p><i>D</i>. <i>discoideum</i> amoebae harboring pSW102 (GFP) were treated for 1 h with different concentrations of (A) racemic LAI-1, (B) 10 μM (<i>R</i>)-LAI-1, (<i>S</i>)-LAI-1, (<i>R</i>)-amino-LAI-1 or (<i>S</i>)-amino-LAI-1, or (C) different concentrations of CAI-1, and cell migration towards folate (1 mM) was monitored in under-agarose assays for 4 h. Graphs depict per cent GFP fluorescence intensity versus migration distance. (D) <i>D</i>. <i>discoideum</i> amoebae harboring pSW102 (GFP) were treated with LAI-1 (10 μM, 1 h). Single cell migration towards folate (1 mM) was monitored in under-agarose assays for 15 min. Motility parameters (forward migration index, FMI; and velocity) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Murine RAW 264.7 macrophages were treated for 1 h with different concentrations of racemic LAI-1, cell migration towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 h, and the cells were stained with Cell Tracker Green BODIPY. Macrophages treated for 1 h with 10 μM LAI-1 were immuno-labeled for (F) α-tubulin (green) or (G) actin (red) and, as a control, the production of cellular α-tubulin or actin was quantified by Western blot. Microtubule fibers per cell were counted along cross-sections (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005307#ppat.1005307.s003" target="_blank">S3 Fig</a>), and the actin architecture was analyzed by quantifying the number of cells displaying cortical actin. The graphs show means and standard deviations of 3 independent experiments (n > 25 (α-tubulin) or > 40 (actin) single cells; Student´s t-test, *<i>p</i> < 0.05, **<i>p</i> < 0.01). Bars (F, G), 5 μm.</p

LAI-1-dependent inhibition of cell migration requires the Cdc42 GEF ARHGEF9.

<p>(A) Confluent cell layers of A549 cells were treated for 2 days with siRNA against the different Cdc42 GEFs or GAPs indicated. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified at 6 different positions per condition using ImageJ software. Means and standard deviations of 3 samples are shown, which are representative of 3 independent experiments (***<i>p</i> < 0.001).</p

LAI-1 reverses Icm/Dot-dependent inhibition of migration by <i>L</i>. <i>pneumophila</i>.

<p>(A) <i>D</i>. <i>discoideum</i> Ax3 amoebae harboring pSW102 (GFP) or (B) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria and treated with different concentrations of LAI-1 (1, 5 and 10 μM) or not. The effect of LAI-1 on migration of amoebae towards folate (1 mM) or macrophages towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 hours. Macrophages were stained with Cell Tracker Green BODIPY. Graphs depict the per cent fluorescence intensity versus migration distance. (C) <i>D</i>. <i>discoideum</i> Ax3 amoebae harboring pSW102 (GFP) or (D) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria and treated with LAI-1 (10 μM, 1 h) or not. Single cell migration towards folate (1 mM) or CCL5 (100 ng/ml) was tracked in an under-agarose assay for 15 min or 1 h, respectively. Motility parameters (forward migration index, FMI, and velocity (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005307#ppat.1005307.s007" target="_blank">S7 Fig</a>)) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria, treated with LAI-1 (10 μM) or not, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (F) The scratch area was quantified at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (C, D, F; means and standard deviations; *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001).</p

Migration inhibition by <i>L</i>. <i>pneumophila</i> is augmented in the absence of Cdc42.

<p>(A) Confluent cell layers of A549 cells were treated with (A) scrambled siRNA or siRNA against (B) Cdc42 or (C) Rac1 for 2 days, left uninfected or infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified after 24 h at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (***<i>p</i> < 0.001). (C) <i>L</i>. <i>pneumophila</i> colocalizes with IQGAP1 and Cdc42. A549 cells were infected (MOI 10, 1 h) with <i>L</i>. <i>pneumophila</i> wild-type or Δ<i>icmT</i> mutant bacteria harboring plasmid pSW001 (DsRed), and the subcellular localization of the scaffold protein (green; FITC) or the small GTPase (green; FITC) was analyzed by confocal microscopy using antibodies against IQGAP1 or Cdc42. Nuclei were stained with DAPI (blue). Bars, 5 μm.</p