Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting...
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doaj-036cd3b1e1b546338192bbbb5c895fe92020-11-24T21:14:36ZengMDPI AGInternational Journal of Molecular Sciences1422-00672018-09-01199269210.3390/ijms19092692ijms19092692Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor AgonistSamantha C. Faber0Anatoly A. Soshilov1Sara Giani Tagliabue2Laura Bonati3Michael S. Denison4Department of Environmental Toxicology, University of California, Davis, CA 95616, USADepartment of Environmental Toxicology, University of California, Davis, CA 95616, USADepartment of Earth and Environmental Sciences, University of Milano-Bicocca, Milan 20126, ItalyDepartment of Earth and Environmental Sciences, University of Milano-Bicocca, Milan 20126, ItalyDepartment of Environmental Toxicology, University of California, Davis, CA 95616, USAThe aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response.http://www.mdpi.com/1422-0067/19/9/2692Ah receptorAhRindirubinTCDDin vitroin silico |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Samantha C. Faber Anatoly A. Soshilov Sara Giani Tagliabue Laura Bonati Michael S. Denison |
spellingShingle |
Samantha C. Faber Anatoly A. Soshilov Sara Giani Tagliabue Laura Bonati Michael S. Denison Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist International Journal of Molecular Sciences Ah receptor AhR indirubin TCDD in vitro in silico |
author_facet |
Samantha C. Faber Anatoly A. Soshilov Sara Giani Tagliabue Laura Bonati Michael S. Denison |
author_sort |
Samantha C. Faber |
title |
Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist |
title_short |
Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist |
title_full |
Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist |
title_fullStr |
Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist |
title_full_unstemmed |
Comparative In Vitro and In Silico Analysis of the Selectivity of Indirubin as a Human Ah Receptor Agonist |
title_sort |
comparative in vitro and in silico analysis of the selectivity of indirubin as a human ah receptor agonist |
publisher |
MDPI AG |
series |
International Journal of Molecular Sciences |
issn |
1422-0067 |
publishDate |
2018-09-01 |
description |
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that modulates gene expression following its binding and activation by structurally diverse chemicals. Species differences in AhR functionality have been observed, with the mouse AhR (mAhR) and human AhR (hAhR) exhibiting significant differences in ligand binding, coactivator recruitment, gene expression and response. While the AhR agonist indirubin (IR) is a more potent activator of hAhR-dependent gene expression than the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), it is a significantly less potent activator of the mAhR. DNA binding analysis confirmed the greater potency/efficacy of IR in stimulating transformation/DNA binding of the hAhR in vitro and domain-swapping experiments demonstrated that the enhanced response to IR was primarily due to the hAhR ligand binding domain (LBD). Site-directed mutagenesis and functional analysis studies revealed that mutation of H326 and A349 in the mAhR LBD to the corresponding residues in the hAhR LBD significantly increased the potency of IR. Since these mutations had no significant effect on ligand binding, these residues likely contribute to an enhanced efficiency of transformation/DNA binding by IR-bound hAhR. Molecular docking to mAhR LBD homology models further elucidated the different roles of the A375V mutation in TCDD and IR binding, as revealed by [3H]TCDD competitive binding results. These results demonstrate the differential binding of structurally diverse ligands within the LBD of a given AhR and confirm that amino acid differences within the LBD of AhRs contribute to significant species differences in ligand response. |
topic |
Ah receptor AhR indirubin TCDD in vitro in silico |
url |
http://www.mdpi.com/1422-0067/19/9/2692 |
work_keys_str_mv |
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