RESUMO
The aryl hydrocarbon receptor (AHR) plays pleiotropic roles in the development and physiology of vertebrates in conjunction with xenobiotic and endogenous ligands. It is best known for mediating the toxic effects of dioxin-like pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). While most vertebrates possess at least one AHR that binds TCDD tightly, amphibian AHRs bind TCDD with very low affinity. Previous analyses of AHRs from Xenopus laevis (a frog; order Anura) and Ambystoma mexicanum (a salamander; order Caudata) identified three amino acid residues in the ligand-binding domain (LBD) that underlie low-affinity binding. In X. laevis AHR1ß, these are A354, A370, and N325. Here we extend the analysis of amphibian AHRs to the caecilian Gymnopis multiplicata, representing the remaining extant amphibian order, Gymnophiona. G. multiplicata AHR groups with the monophyletic vertebrate AHR/AHR1 clade. The LBD includes all three signature residues of low TCDD affinity, and a structural homology model suggests that its architecture closely resembles those of other amphibians. In transactivation assays, the EC50 for reporter gene induction by TCDD was 17.17 nM, comparable to X. laevis AhR1ß (26.23 nM) and Ambystoma AHR (34.09 nM) and dramatically higher than mouse AhR (0.13 nM), a trend generally reflected in direct measures of TCDD binding. These shared properties distinguish amphibian AHRs from the high-affinity proteins typical of both vertebrate groups that diverged earlier (teleost fish) and those that appeared more recently (other tetrapods). These findings suggest the hypothesis that AHRs with low TCDD affinity represent a characteristic that evolved in a common ancestor of all three extant amphibian groups.
Assuntos
Ambystoma mexicanum/metabolismo , Dibenzodioxinas Policloradas/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Xenopus laevis/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ligantes , Filogenia , Dibenzodioxinas Policloradas/química , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Homologia de SequênciaRESUMO
1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ) are clinically promising biologically active chemicals that have been shown to stimulate the aryl hydrocarbon receptor (AhR) signaling pathway, but whether they are direct or indirect ligands or activate the AhR in a ligand-independent manner is unknown. Given the structural diversity of AhR ligands, multiple mechanisms of AhR activation of gene expression, and species differences in AhR ligand binding and response, we examined the ability of 1,2-NQ and 1,4-NQ to bind to and activate the mouse and human AhRs using a series of in vitro AhR-specific bioassays and in silico modeling techniques. Both NQs induced AhR-dependent gene expression in mouse and human hepatoma cells, but were more potent and efficacious in human cells. 1,2-NQ and 1,4-NQ stimulated AhR transformation and DNA binding in vitro and was inhibited by AhR antagonists. Ligand binding analysis confirmed the ability of 1,2-NQ and 1,4-NQ to competitively bind to the AhR ligand binding cavity and the molecular determinants for interactions were predicted by molecular modeling methods. NQs were shown to bind distinctly differently from that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and differences were also observed between species. Mutation of amino acid residues (F289, M334, and M342) involved in critical NQ:AhR binding interactions, decreased NQ- and AhR-dependent gene expression, consistent with a role for these residues in binding and activation of the AhR by NQs. These studies provide insights into the molecular mechanism of action of NQs and contribute to the development of emerging NQ-based therapeutics.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Naftoquinonas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Ligação Competitiva , Células COS , Linhagem Celular , Chlorocebus aethiops , Citocromo P-450 CYP1A1/genética , DNA/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Naftoquinonas/metabolismo , Dibenzodioxinas Policloradas/farmacologia , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genética , Especificidade da EspécieRESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Receptores de Hidrocarboneto Arílico/agonistas , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Simulação por Computador , Humanos , Técnicas In Vitro , Indóis/farmacologia , Camundongos , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Dibenzodioxinas Policloradas/farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Receptores de Hidrocarboneto Arílico/genética , Especificidade da EspécieRESUMO
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor whose activity can be modulated by polyphenols, such as curcumin. AhR and curcumin have evolutionarily conserved effects on aging. Here, we investigated whether and how the AhR mediates the anti-aging effects of curcumin across species. Using a combination of in vivo, in vitro, and in silico analyses, we demonstrated that curcumin has AhR-dependent or -independent effects in a context-specific manner. We found that in Caenorhabditis elegans, AhR mediates curcumin-induced lifespan extension, most likely through a ligand-independent inhibitory mechanism related to its antioxidant activity. Curcumin also showed AhR-independent anti-aging activities, such as protection against aggregation-prone proteins and oxidative stress in C. elegans and promotion of the migratory capacity of human primary endothelial cells. These AhR-independent effects are largely mediated by the Nrf2/SKN-1 pathway.
RESUMO
The Ah receptor (AhR) is a ligand-dependent transcription factor belonging to the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) superfamily. Binding to and activation of the AhR by a variety of chemicals results in the induction of expression of diverse genes and production of a broad spectrum of biological and toxic effects. The AhR also plays important roles in several physiological responses, which has led it to become a novel target for the development of therapeutic drugs. Differences in the interactions of various ligands within the AhR ligand binding domain (LBD) may contribute to differential modulation of AhR functionality. We combined computational and experimental analyses to investigate the binding modes of a group of chemicals representative of major classes of AhR ligands. On the basis of a novel computational approach for molecular docking to the homology model of the AhR LBD that includes the receptor flexibility, we predicted specific residues within the AhR binding cavity that play a critical role in binding of three distinct groups of chemicals. The prediction was validated by site-directed mutagenesis and evaluation of the relative ligand binding affinities for the mutant AhRs. These results provide an avenue for understanding ligand modulation of the AhR functionality and for rational drug design.
Assuntos
Modelos Moleculares , Receptores de Hidrocarboneto Arílico/metabolismo , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica/fisiologiaRESUMO
The Pregnane X Receptor (PXR) is a ligand-activated transcription factor belonging to the nuclear receptor family. PXR can bind diverse drugs and environmental toxicants with different binding modes, making it an intriguing target for drug discovery. Here we investigated the binding mechanism of the SR12813 ligand to elucidate the significant steps, from the ligand entrance pathway into the binding cavity, to the ligand-induced conformational changes, and to the exploration of its alternative binding geometries. We used the advanced Molecular Dynamics-based methods implemented in the BiKi suite and developed specific methodological approaches to overcome the complexity induced by the buried and flexible binding cavity. The adopted methods provided a full dynamic description of the binding event and allowed rationalization of the observed multiple binding modes. These results suggest that the same approach could be exploited for the study of other binding processes with similar characteristics.