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AHR2 mutant reveals functional diversity of aryl hydrocarbon receptors in zebrafish.

Goodale BC, La Du JK, Bisson WH, Janszen DB, Waters KM, Tanguay RL - PLoS ONE (2012)

Bottom Line: Homology modeling, however, predicted a ligand binding conformation of AHR1A with leflunomide.AHR1A-dependent CYP1A immunohistochemical expression in the liver provided in vivo confirmation of the in silico docking studies.The ahr2(hu3335) functional knockout line expands the experimental power of zebrafish to unravel the role of the AHR during development, as well as highlights potential activity of the other AHR paralogues in ligand-specific toxicological responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon, United States of America.

ABSTRACT
The aryl hydrocarbon receptor (AHR) is well known for mediating the toxic effects of TCDD and has been a subject of intense research for over 30 years. Current investigations continue to uncover its endogenous and regulatory roles in a wide variety of cellular and molecular signaling processes. A zebrafish line with a mutation in ahr2 (ahr2(hu3335)), encoding the AHR paralogue responsible for mediating TCDD toxicity in zebrafish, was developed via Targeting Induced Local Lesions IN Genomes (TILLING) and predicted to express a non-functional AHR2 protein. We characterized AHR activity in the mutant line using TCDD and leflunomide as toxicological probes to investigate function, ligand binding and CYP1A induction patterns of paralogues AHR2, AHR1A and AHR1B. By evaluating TCDD-induced developmental toxicity, mRNA expression changes and CYP1A protein in the AHR2 mutant line, we determined that ahr2(hu3335) zebrafish are functionally . In silico modeling predicted differential binding of TCDD and leflunomide to the AHR paralogues. AHR1A is considered a non-functional pseudogene as it does not bind TCCD or mediate in vivo TCDD toxicity. Homology modeling, however, predicted a ligand binding conformation of AHR1A with leflunomide. AHR1A-dependent CYP1A immunohistochemical expression in the liver provided in vivo confirmation of the in silico docking studies. The ahr2(hu3335) functional knockout line expands the experimental power of zebrafish to unravel the role of the AHR during development, as well as highlights potential activity of the other AHR paralogues in ligand-specific toxicological responses.

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Molecular docking of TCDD and Leflunomide in zebrafish AHR isoforms.A) TCDD docking orientation in zebrafish AHR2- and B) AHR1B-LBD homology model binding pocket (ICM v3.5-1n, Molsoft). C) Leflunomide docking orientation into AHR2-, D) AHR1B- and E) AHR1A homology model binding pockets. The residues are displayed as sticks and colored by atom type with the carbon atoms in green. The protein backbone is displayed as ribbon and colored by secondary structure. The ligand is displayed as sticks and colored by atom type with carbon atoms in orange (A, C), magenta (B, D) and yellow (E). H-bonds are represented by black dashed lines.
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pone-0029346-g005: Molecular docking of TCDD and Leflunomide in zebrafish AHR isoforms.A) TCDD docking orientation in zebrafish AHR2- and B) AHR1B-LBD homology model binding pocket (ICM v3.5-1n, Molsoft). C) Leflunomide docking orientation into AHR2-, D) AHR1B- and E) AHR1A homology model binding pockets. The residues are displayed as sticks and colored by atom type with the carbon atoms in green. The protein backbone is displayed as ribbon and colored by secondary structure. The ligand is displayed as sticks and colored by atom type with carbon atoms in orange (A, C), magenta (B, D) and yellow (E). H-bonds are represented by black dashed lines.

Mentions: We recently reported a homology model that has been used to predict binding affinity of potential ligands to the human, mouse and zebrafish AHRs [39]. In order to investigate differential function of the zebrafish AHR paralogues, we tested TCDD and a known AHR ligand with a non-classical structure, leflunomide, in a series of molecular docking studies. Sequence alignment of the mouse and zebrafish AHR-PASB domains produced identities of 65.1% (zfAHR1A), 78.5% (zfAHR1B) and 70.5% (zfAHR2). High similarity between the three isoforms at the primary and predicted tertiary structural levels was also noted, with 74.3% (AHR2/1B) and 69.9% (AHR1B/1A) identity. TCDD and leflunomide were docked into zebrafish AHR1A-, AHR1B-, and AHR2-LBD homology models. TCDD docked in AHR2 and AHR1B with predicted binding energies of −3.97 kcal/mol and −4.86 kcal/mol, respectively, but was unable to dock in AHR1A (Table 2, Figure 5A,B). Leflunomide was also able dock in AHR2 and AHR1B, with predicted binding energies of −2.13 kcal/mol and −1.97 kcal/mol, respectively (Table 2, Figure 5C,D). Interestingly, in contrast to TCDD, leflunomide docked into AHR1A, but in a unique orientation [17] (Figure 5E).


AHR2 mutant reveals functional diversity of aryl hydrocarbon receptors in zebrafish.

Goodale BC, La Du JK, Bisson WH, Janszen DB, Waters KM, Tanguay RL - PLoS ONE (2012)

Molecular docking of TCDD and Leflunomide in zebrafish AHR isoforms.A) TCDD docking orientation in zebrafish AHR2- and B) AHR1B-LBD homology model binding pocket (ICM v3.5-1n, Molsoft). C) Leflunomide docking orientation into AHR2-, D) AHR1B- and E) AHR1A homology model binding pockets. The residues are displayed as sticks and colored by atom type with the carbon atoms in green. The protein backbone is displayed as ribbon and colored by secondary structure. The ligand is displayed as sticks and colored by atom type with carbon atoms in orange (A, C), magenta (B, D) and yellow (E). H-bonds are represented by black dashed lines.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3252317&req=5

pone-0029346-g005: Molecular docking of TCDD and Leflunomide in zebrafish AHR isoforms.A) TCDD docking orientation in zebrafish AHR2- and B) AHR1B-LBD homology model binding pocket (ICM v3.5-1n, Molsoft). C) Leflunomide docking orientation into AHR2-, D) AHR1B- and E) AHR1A homology model binding pockets. The residues are displayed as sticks and colored by atom type with the carbon atoms in green. The protein backbone is displayed as ribbon and colored by secondary structure. The ligand is displayed as sticks and colored by atom type with carbon atoms in orange (A, C), magenta (B, D) and yellow (E). H-bonds are represented by black dashed lines.
Mentions: We recently reported a homology model that has been used to predict binding affinity of potential ligands to the human, mouse and zebrafish AHRs [39]. In order to investigate differential function of the zebrafish AHR paralogues, we tested TCDD and a known AHR ligand with a non-classical structure, leflunomide, in a series of molecular docking studies. Sequence alignment of the mouse and zebrafish AHR-PASB domains produced identities of 65.1% (zfAHR1A), 78.5% (zfAHR1B) and 70.5% (zfAHR2). High similarity between the three isoforms at the primary and predicted tertiary structural levels was also noted, with 74.3% (AHR2/1B) and 69.9% (AHR1B/1A) identity. TCDD and leflunomide were docked into zebrafish AHR1A-, AHR1B-, and AHR2-LBD homology models. TCDD docked in AHR2 and AHR1B with predicted binding energies of −3.97 kcal/mol and −4.86 kcal/mol, respectively, but was unable to dock in AHR1A (Table 2, Figure 5A,B). Leflunomide was also able dock in AHR2 and AHR1B, with predicted binding energies of −2.13 kcal/mol and −1.97 kcal/mol, respectively (Table 2, Figure 5C,D). Interestingly, in contrast to TCDD, leflunomide docked into AHR1A, but in a unique orientation [17] (Figure 5E).

Bottom Line: Homology modeling, however, predicted a ligand binding conformation of AHR1A with leflunomide.AHR1A-dependent CYP1A immunohistochemical expression in the liver provided in vivo confirmation of the in silico docking studies.The ahr2(hu3335) functional knockout line expands the experimental power of zebrafish to unravel the role of the AHR during development, as well as highlights potential activity of the other AHR paralogues in ligand-specific toxicological responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, Oregon, United States of America.

ABSTRACT
The aryl hydrocarbon receptor (AHR) is well known for mediating the toxic effects of TCDD and has been a subject of intense research for over 30 years. Current investigations continue to uncover its endogenous and regulatory roles in a wide variety of cellular and molecular signaling processes. A zebrafish line with a mutation in ahr2 (ahr2(hu3335)), encoding the AHR paralogue responsible for mediating TCDD toxicity in zebrafish, was developed via Targeting Induced Local Lesions IN Genomes (TILLING) and predicted to express a non-functional AHR2 protein. We characterized AHR activity in the mutant line using TCDD and leflunomide as toxicological probes to investigate function, ligand binding and CYP1A induction patterns of paralogues AHR2, AHR1A and AHR1B. By evaluating TCDD-induced developmental toxicity, mRNA expression changes and CYP1A protein in the AHR2 mutant line, we determined that ahr2(hu3335) zebrafish are functionally . In silico modeling predicted differential binding of TCDD and leflunomide to the AHR paralogues. AHR1A is considered a non-functional pseudogene as it does not bind TCCD or mediate in vivo TCDD toxicity. Homology modeling, however, predicted a ligand binding conformation of AHR1A with leflunomide. AHR1A-dependent CYP1A immunohistochemical expression in the liver provided in vivo confirmation of the in silico docking studies. The ahr2(hu3335) functional knockout line expands the experimental power of zebrafish to unravel the role of the AHR during development, as well as highlights potential activity of the other AHR paralogues in ligand-specific toxicological responses.

Show MeSH
Related in: MedlinePlus