Mathäs M et al. (2014), Structural and functional similarity of amphibi...

XB-ART-48958

PLoS One 2014 Jan 01;95:e96263. doi: 10.1371/journal.pone.0096263.

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Structural and functional similarity of amphibian constitutive androstane receptor with mammalian pregnane X receptor.

Mathäs M , Nusshag C , Nuβhag C , Burk O , Gödtel-Armbrust U , Herlyn H , Wojnowski L , Windshügel B .

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The nuclear receptors and xenosensors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2) induce the expression of xenobiotic metabolizing enzymes and transporters, which also affects various endobiotics. While human and mouse CAR feature a high basal activity and low induction upon ligand exposure, we recently identified two constitutive androstane receptors in Xenopus laevis (xlCARá and â) that possess PXR-like characteristics such as low basal activity and activation in response to structurally diverse compounds. Using a set of complementary computational and biochemical approaches we provide evidence for xlCARá being the structural and functional counterpart of mammalian PXR. A three-dimensional model of the xlCARá ligand-binding domain (LBD) reveals a human PXR-like L-shaped ligand binding pocket with a larger volume than the binding pockets in human and murine CAR. The shape and amino acid composition of the ligand-binding pocket of xlCAR suggests PXR-like binding of chemically diverse ligands which was confirmed by biochemical methods. Similarly to PXR, xlCARá possesses a flexible helix 11'. Modest increase in the recruitment of coactivator PGC-1á may contribute to the enhanced basal activity of three gain-of-function xlCARá mutants humanizing key LBD amino acid residues. xlCARá and PXR appear to constitute an example of convergent evolution.

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Species referenced: Xenopus laevis
Genes referenced: bpi.4 ca1 lgals4.2 ncoa1 pgc src

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	Figure 2. Comparison of ligand-binding domain structures.The course of the protein backbone for hsPXR (A, PDB code 1nrl) and hsCAR (B, PDB code 1xv9) x-ray crystal structures, as well as the xlCARα homology model (C) is displayed using ribbons. The shape of the ligand-binding pocket is indicated using a surface representation. Hydrophobic areas of the LBP are represented in brown, hydrophilic areas are colored in blue.
	Figure 3. Basal activity of xlCARα mutants and their constitutive interaction with RXRα and coactivators.(A) LS174T cells were co-transfected with the DR4-TK promoter reporter gene plasmid and expression plasmids encoding the indicated xlCARα mutants. Data are presented as % of normalized activity of wild type xlCARα, which was set as 100% and is indicated as a red line. Bars show means ±SEM (n≥8 independent experiments). Statistically significant differences to wild type xlCARα, as determined by one-sample t-tests (corrected for multiple testing by the method of Bonferroni) are indicated by asterisks. (B–D) Mammalian two-hybrid assays were used to analyze the interaction of xlCARα mutants with RXRα (B), SRC-1 (C) and PGC-1α (D). To this end COS7 cells were co-transfected with the GAL4-dependent pGL3G5 reporter, and expression plasmids encoding fusions of VP16-AD and the respective LBD of the indicated xlCARα mutants together with expression plasmids encoding fusions of GAL4-DBD/RXRα-LBD (B), or GAL4-DBD/SRC1-RID (C), or GAL4-DBD/PGC-1α-RID (D). For (B) bars show means ± SEM (n = 6 independent experiments), with activity of cells transfected with empty vector pVP16-AD set as 1. Statistically significant differences to the activity of wild type xlCARα, as identified by one-way ANOVA with Dunnett's multiple comparisons test are indicated by asterisks. For (C) and (D) bars show means ± SEM (n = 9 independent experiments), with activity of cells transfected with the combination of both empty expression plasmids pVP16-AD and pM set as 1. Statistically significant differences to the activity of pVP16-AD and the respective GAL4-DBD/coactivator RID, as identified by two-way ANOVA with Dunnett's multiple comparisons test, are indicated by asterisks. , P<0.05; , P<0.01; **, P<0.001, no asterisks indicate no differences. Red vertical lines in C and D indicate the activity of the empty vector pVP16-AD.
	Figure 4. The importance of the H10/11–H12 region for X.laevis and human CAR function.Change in receptor basal activity upon deletion or insertion of two amino acids in human and Xenopus CAR as well as introduction of the helix-breaking amino acid proline. LS174T cells were co-transfected with the DR4-TK promoter reporter gene plasmid and expression plasmids encoding the indicated CAR proteins. Bars show means ± SEM, with activity of cells transfected with empty expression vector pcDNA3 designated as 1. Statistically significant differences to the activity of the respective wild type CAR were analyzed by one-way ANOVA with Dunnett's multiple comparisons test and indicated by asterisks. , P<0.05; , P<0.01; **, P<0.001.
	Figure 5. Binding modes of xlCARα agonists within the receptor ligand-binding pocket.Favorable docking poses for artemisinin (A), fenofibrate (B) and pregnanedione (C) within the ligand-binding pocket (LBP) of xlCARα were determined using docking program GOLD. Selected amino acids of the LBP are shown as capped sticks while ligands are displayed in ball and stick representation. Hydrogen bonds between ligands and protein are shown as dotted lines.
	Figure 6. Constitutive interaction and ligand-dependent induction of the interaction of xlCARα mutants with coactivator SRC-1.CARLA assays were performed using bacterially expressed GST-SRC-1 RID protein and in vitro translated 35S-labeled xlCARα wild type or mutant proteins or human CAR1 protein (hsCAR1), respectively, in the presence of the indicated concentrations of pregnanedione or solvent only (DMSO). (A) The results of representative experiments with xlCARα wild type and mutant proteins are shown. (B,C) Dose response analysis of ligand-dependent induction of SRC-1 interaction of xlCARα mutants (B) and human CAR1 (C), with graphs showing means ± SD (n = 3 independent experiments) of fold induction by respective concentrations of pregnanedione. Activity in the presence of DMSO only was designated as 1. In the upper part of (C), a representative experiment with human CAR1 is shown. Data were analyzed by two-way ANOVA with Dunnett's multiple comparisons test (B) or one sample t-test (C). Significant differences to wild type xlCARα are shown by asterisks. The boxed asterisks indicate significant difference for all mutants at the respective dose. (D) Columns show means ± SD (n = 3 independent experiments) of the constitutive interaction with SRC-1 in the absence of ligand. Data were analyzed by one-way ANOVA with Dunnett's multiple comparisons test. Significant difference to wild type xlCARα is shown by asterisks. ***, P<0.001.
	Figure 7. Dose-reponse analysis of ligand-dependent activities of xlCARα mutants.HepG2 cells were co-transfected with expression plasmids encoding wild type xlCARα or the indicated mutants and DR4-TK promoter reporter gene and treated with pregnanedione as indicated. Graphs show means ± SD (n≥3 independent experiments) of fold induction by respective concentrations of pregnanedione, with activity in the presence of solvent DMSO only set as 1. (A) presents the dose response curves of xlCARα proteins with mutations of amino acid residues contacting pregnanedione via vdW interactions, whereas (B) presents the respective dose response curves of xlCARα proteins with mutations of amino acid residues contacting pregnanedione by H-bonds, each in comparison to the same dose response curve of wild type xl CARα. Data were analyzed by two-way ANOVA with Dunnett's multiple comparisons test. Significant differences to wild type xlCARα are shown by asterisks, whereby colors refer to the respective mutants. Black asterisks in a box indicate significant difference for all mutants at the respective dose. , P<0.01; *, P<0.001.
	Figure 1. Role of the H1-H3 interhelical domain for X. laevis CARα and human PXR activity.Comparison of basal (A) and ligand-dependent activities (B) of wildtype xlCARα (xlCARα) and xlCARα with deletion of amino acids 186–229 (44AA-Del xlCARα). LS174T cells were co-transfected with expression plasmids encoding the indicated proteins and the CYP3A4 enhancer/promoter reporter gene plasmid. Comparison of constitutive (C) and ligand-dependent (D) activities of wildtype hsPXR (hsPXR) and its mutant missing AS 182 to 233 (del hsPXR). LS174T cells were co-transfected with respective expression plasmids and a Cyp2B10 promoter element driving luciferase expression. Ligands were used at 10 µM with the exception of artemisinin (100 µM), lovastatin (30 µM) and phenobarbital (1 mM). Data are presented as means ± SEM (n = 6 independent experiments), with activity in the presence of empty expression vector pcDNA3 only (A,C) or of treatment with vehicle DMSO only (B,D) set as 1. Statistically significant differences between wild type and mutant proteins, as determined by t-test (A,C) or one-way ANOVA with Dunnett's multiple comparisons test (B,D), are indicated by asterisks. , P<0.05; , P<0.01; **, P<0.001, no asterisks indicate no differences.

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	Figure 2. Comparison of ligand-binding domain structures.The course of the protein backbone for hsPXR (A, PDB code 1nrl) and hsCAR (B, PDB code 1xv9) x-ray crystal structures, as well as the xlCARα homology model (C) is displayed using ribbons. The shape of the ligand-binding pocket is indicated using a surface representation. Hydrophobic areas of the LBP are represented in brown, hydrophilic areas are colored in blue.
	Figure 3. Basal activity of xlCARα mutants and their constitutive interaction with RXRα and coactivators.(A) LS174T cells were co-transfected with the DR4-TK promoter reporter gene plasmid and expression plasmids encoding the indicated xlCARα mutants. Data are presented as % of normalized activity of wild type xlCARα, which was set as 100% and is indicated as a red line. Bars show means ±SEM (n≥8 independent experiments). Statistically significant differences to wild type xlCARα, as determined by one-sample t-tests (corrected for multiple testing by the method of Bonferroni) are indicated by asterisks. (B–D) Mammalian two-hybrid assays were used to analyze the interaction of xlCARα mutants with RXRα (B), SRC-1 (C) and PGC-1α (D). To this end COS7 cells were co-transfected with the GAL4-dependent pGL3G5 reporter, and expression plasmids encoding fusions of VP16-AD and the respective LBD of the indicated xlCARα mutants together with expression plasmids encoding fusions of GAL4-DBD/RXRα-LBD (B), or GAL4-DBD/SRC1-RID (C), or GAL4-DBD/PGC-1α-RID (D). For (B) bars show means ± SEM (n = 6 independent experiments), with activity of cells transfected with empty vector pVP16-AD set as 1. Statistically significant differences to the activity of wild type xlCARα, as identified by one-way ANOVA with Dunnett's multiple comparisons test are indicated by asterisks. For (C) and (D) bars show means ± SEM (n = 9 independent experiments), with activity of cells transfected with the combination of both empty expression plasmids pVP16-AD and pM set as 1. Statistically significant differences to the activity of pVP16-AD and the respective GAL4-DBD/coactivator RID, as identified by two-way ANOVA with Dunnett's multiple comparisons test, are indicated by asterisks. , P<0.05; , P<0.01; **, P<0.001, no asterisks indicate no differences. Red vertical lines in C and D indicate the activity of the empty vector pVP16-AD.
	Figure 4. The importance of the H10/11–H12 region for X.laevis and human CAR function.Change in receptor basal activity upon deletion or insertion of two amino acids in human and Xenopus CAR as well as introduction of the helix-breaking amino acid proline. LS174T cells were co-transfected with the DR4-TK promoter reporter gene plasmid and expression plasmids encoding the indicated CAR proteins. Bars show means ± SEM, with activity of cells transfected with empty expression vector pcDNA3 designated as 1. Statistically significant differences to the activity of the respective wild type CAR were analyzed by one-way ANOVA with Dunnett's multiple comparisons test and indicated by asterisks. , P<0.05; , P<0.01; **, P<0.001.
	Figure 5. Binding modes of xlCARα agonists within the receptor ligand-binding pocket.Favorable docking poses for artemisinin (A), fenofibrate (B) and pregnanedione (C) within the ligand-binding pocket (LBP) of xlCARα were determined using docking program GOLD. Selected amino acids of the LBP are shown as capped sticks while ligands are displayed in ball and stick representation. Hydrogen bonds between ligands and protein are shown as dotted lines.
	Figure 6. Constitutive interaction and ligand-dependent induction of the interaction of xlCARα mutants with coactivator SRC-1.CARLA assays were performed using bacterially expressed GST-SRC-1 RID protein and in vitro translated 35S-labeled xlCARα wild type or mutant proteins or human CAR1 protein (hsCAR1), respectively, in the presence of the indicated concentrations of pregnanedione or solvent only (DMSO). (A) The results of representative experiments with xlCARα wild type and mutant proteins are shown. (B,C) Dose response analysis of ligand-dependent induction of SRC-1 interaction of xlCARα mutants (B) and human CAR1 (C), with graphs showing means ± SD (n = 3 independent experiments) of fold induction by respective concentrations of pregnanedione. Activity in the presence of DMSO only was designated as 1. In the upper part of (C), a representative experiment with human CAR1 is shown. Data were analyzed by two-way ANOVA with Dunnett's multiple comparisons test (B) or one sample t-test (C). Significant differences to wild type xlCARα are shown by asterisks. The boxed asterisks indicate significant difference for all mutants at the respective dose. (D) Columns show means ± SD (n = 3 independent experiments) of the constitutive interaction with SRC-1 in the absence of ligand. Data were analyzed by one-way ANOVA with Dunnett's multiple comparisons test. Significant difference to wild type xlCARα is shown by asterisks. ***, P<0.001.
	Figure 7. Dose-reponse analysis of ligand-dependent activities of xlCARα mutants.HepG2 cells were co-transfected with expression plasmids encoding wild type xlCARα or the indicated mutants and DR4-TK promoter reporter gene and treated with pregnanedione as indicated. Graphs show means ± SD (n≥3 independent experiments) of fold induction by respective concentrations of pregnanedione, with activity in the presence of solvent DMSO only set as 1. (A) presents the dose response curves of xlCARα proteins with mutations of amino acid residues contacting pregnanedione via vdW interactions, whereas (B) presents the respective dose response curves of xlCARα proteins with mutations of amino acid residues contacting pregnanedione by H-bonds, each in comparison to the same dose response curve of wild type xl CARα. Data were analyzed by two-way ANOVA with Dunnett's multiple comparisons test. Significant differences to wild type xlCARα are shown by asterisks, whereby colors refer to the respective mutants. Black asterisks in a box indicate significant difference for all mutants at the respective dose. , P<0.01; *, P<0.001.
	Figure 1. Role of the H1-H3 interhelical domain for X. laevis CARα and human PXR activity.Comparison of basal (A) and ligand-dependent activities (B) of wildtype xlCARα (xlCARα) and xlCARα with deletion of amino acids 186–229 (44AA-Del xlCARα). LS174T cells were co-transfected with expression plasmids encoding the indicated proteins and the CYP3A4 enhancer/promoter reporter gene plasmid. Comparison of constitutive (C) and ligand-dependent (D) activities of wildtype hsPXR (hsPXR) and its mutant missing AS 182 to 233 (del hsPXR). LS174T cells were co-transfected with respective expression plasmids and a Cyp2B10 promoter element driving luciferase expression. Ligands were used at 10 µM with the exception of artemisinin (100 µM), lovastatin (30 µM) and phenobarbital (1 mM). Data are presented as means ± SEM (n = 6 independent experiments), with activity in the presence of empty expression vector pcDNA3 only (A,C) or of treatment with vehicle DMSO only (B,D) set as 1. Statistically significant differences between wild type and mutant proteins, as determined by t-test (A,C) or one-way ANOVA with Dunnett's multiple comparisons test (B,D), are indicated by asterisks. , P<0.05; , P<0.01; **, P<0.001, no asterisks indicate no differences.