AGU Ocean Sciences meeting 2010

<p>Slides from a 2010 oral presentation given at the AGU Ocean Sciences meeting in Portland, Oregon. </p

Additional file 1: of MicrO: an ontology of phenotypic and metabolic characters, assays, and culture media found in prokaryotic taxonomic descriptions

Supplemental Figures (Figures S1-S6) and Tables (Tables S1 and S2). (DOCX 1785 kb

Effect of BMS-192364 in Combination with Other Modulators of Calcium Signaling

<div><p>The graphs display fluorescence intensity measurements for HEK293 cells preloaded with Fluo-4 then stimulated with the muscarinic GPCR agonist carbachol at 100 μM. Five baseline fluorescence measurements were taken prior to the injection of carbachol. Where indicated, BMS-195270 or BMS-192364 (100 μM) were added 15 min prior to the carbachol stimulation. The timing of carbachol addition is indicated by a black arrowhead.</p><p>(A) Where indicated, cells were pre-incubated for 15 min with the calcium channel blocker niguldipine (100 μM).</p><p>(B) Where indicated, cells were pre-incubated for 24 h with the G-protein antagonist pertussis toxin (150 ng/ml).</p><p>(C) The treated cells are overexpressing the G-αq mutant allele G188S, which is known to be insensitive to RGS GAP activity.</p></div

Analysis of Molecular Signaling Events Controlling Muscular Contraction in C. elegans

<p>The diagram shows the molecular components of the signaling pathways downstream of the muscarinic GPCRs in <i>C. elegans</i>. Alleles of genes encoding proteins in the pathway were tested for their effect on the Egl-d phenotype caused by treatment with BMS-192364. For the pathway members indicated by stars, certain alleles altered the response to treatment with BMS-192364. Specifically, resistance to the Egl-d phenotype was conferred by two gain-of-function alleles of <i>egl-19</i> and one of <i>egl-30,</i> and by two loss-of-function alleles of <i>eat-16</i> and one of <i>goa-1</i>.</p

Effect of Small Molecules on Wild-Type and Mutant C. elegans

<div><p>(A) The gonad/vulval region of wild-type worms is shown. In the left panel, black arrows indicate the normal, organized array of early stage eggs. The right panel shows a worm treated with BMS-192364 at 0.3 mM. The white arrows indicate late stage eggs that have been retained in the gonad.</p><p>(B) Dose-response curve for BMS-192364, showing effect on egg laying in C. elegans. The percentage of worms displaying an Egl-d phenotype was determined by counting the number of “commas” contained within the animal.</p><p>(C) Quantification of the Egl-d phenotype in four C. elegans mutant strains—<i>ep271, ep272, ep273,</i> and <i>ep275—</i>that were identified in a screen for resistance to the small molecule. Black bars, no treatment. Grey bars, worms treated with BMS-192364 at 0.4 mM.</p><p>(D) Table showing identity of the affected gene in <i>C. elegans–</i>resistant mutant strains, the amino acid changes, and predicted effect on protein function.</p></div

Amino Acid Substitutions in Mutants of the Yeast G-αq Protein Gpa1

<p>A theoretical three-dimensional structure of the yeast Gpa1 G-α protein in complex with the Ste4 protein (G-β) is shown. The position of four amino acid substitutions with phenotypes of interest is indicated by circles. Two alpha helices are indicated by yellow highlighting of the protein backbone. In higher eukaryotes, these helices are considered to form the interface with G-αq downstream effector proteins. Three mutations affecting adaptation to mating pheromone lie on this face: E355K and E364K both hyper-adapt while the M362I allele described in this work is hypo-adaptive. For reference, the position of a mutation affecting sensitivity to RGS GAP activity, G302S, is also shown.</p

The Yeast <i>gpa1-M362I</i> Mutant Allele Causes a Hypo-Adaptation Phenotype

<div><p>Images show the growth of a monolayer of yeast cells around a paper disc containing alpha factor, the peptide ligand for the Ste3 GPCR. A zone of growth inhibition is visible as a “halo” around each disc.</p><p>(A) Yeast strain contains a wild-type G-αq gene <i>(GPA1)</i> and has a chromosomal deletion of the <i>SST2</i> gene, encoding an RGS protein.</p><p>(B) Yeast strain contains a wild-type G-αq gene <i>(GPA1)</i> and a chromosomal deletion of the <i>SST2</i> gene, but carries wild-type <i>SST2</i> on a plasmid.</p><p>(C) Yeast strain contains a mutant G-αq gene <i>(gpa1-M362I)</i> and has a chromosomal deletion of the <i>SST2</i> gene, encoding an RGS protein.</p><p>(D) Yeast strain contains a mutant G-αq gene <i>(gpa1-M362I)</i> and a chromosomal deletion of the <i>SST2</i> gene, but carries wild-type <i>SST2</i> on a plasmid.</p></div

Models for Mechanism of Action

<p>Four models of small-molecule action are presented. In model 1, the small-molecule acts directly and uniquely as an antagonist of G-αq. In model 2 the small-molecule acts directly and uniquely as an agonist of the RGS protein's GAP activity. In models 3 and 4 (our preferred models), the small molecule interacts with both the RGS protein and G-αq, leading to an increased affinity of RGS for the complex and/or an “abortive” complex (failure of G-αq to recycle). All models lead to reduction of the GPCR signal through the activated G-αq.</p

Effect of BMS Small Molecules in Ex Vivo Whole-Bladder Assays

<div><p>Bladder pressure was monitored during saline infusion in the presence of BMS-195270 or vehicle. A typical cystometric curve shows three phases: a small rapid rise in intravesical pressure followed by a plateau phase and then a final sharp increase in pressure.</p><p>(A) Bladder pressure following treatment with BMS-195270 (3 μM; <i>n</i> = 5 bladders). The average pressure at discrete infusion volumes is shown. Significant differences in pressure relative to control (<i>p</i> < 0.05) are indicated by asterisks.</p><p>(B) Bladder pressure following treatment with vehicle (<i>n</i> = 12 bladders). The average pressure at discrete infusion volumes is shown. No difference in pressure relative to control was seen.</p><p>(C) Representative trace from vehicle-treated bladder, showing magnitude of spontaneous contractions developed during saline infusion.</p><p>(D) Representative trace from bladder treated with BMS-195270 (3 μM), showing reduction in frequency and magnitude of spontaneous contractions developed during saline infusion.</p></div

Effect of Compounds on Agonist-Induced Calcium Flux

<div><p>(A) The graph displays fluorescence intensity measurements for HEK293 cells preloaded with Fluo-4. Five baseline fluorescence measurements were taken prior to the injection of the muscarinic GPCR agonist carbachol. The timing of agonist addition is indicated by a black arrowhead. Measurements were performed in the presence of vehicle or the BMS small molecules indicated, at 100 μM.</p><p>(B) Dose-response analysis for the effect of compounds on carbachol-stimulated calcium flux. White squares, BMS-195270; EC₅₀ 2 μM. Black squares, BMS-192364; EC₅₀ 9 μM.</p><p>(C) The graph displays fluorescence intensity measurements for primary smooth muscle cells preloaded with Fluo-4. Five baseline fluorescence measurements were taken prior to the injection of the GPCR agonist histamine. The timing of agonist addition is indicated by a black arrowhead. Measurements were performed in the presence of vehicle or BMS-192364 (100 μM).</p></div