Secretory expression of thyroid hormone receptor using transgenic silkworms and its DNA binding activity.

Silkworms are economically important insects that have the ability to produce large amounts of silk. They have mass breeding methods and silk glands, which are specialized tissues that secrete silk fibroin and sericin. Thus, the production of recombinant proteins in a transgenic silkworm system is a promising approach. We developed a silkworm, Bombyx mori, as a host expression insect for recombinant proteins and successfully produced different proteins including antibodies, glycoproteins, and membrane receptors. The thyroid hormone receptor (TR) is a regulatory factor for many physiological phenomena. It is a lipophilic protein that has DNA-binding and ligand-binding domains. Based on our previous experiences, it was inferred that the recombinant TR easily formed aggregates and precipitates which is potentially due to an unstructured hinge domain. We applied the silkworm expression system to produce mice TRß1 that was fused with glutathione S-transferase. Using 160 larvae, the yield of the recombinant GST-TRß was approximately 4 mg, and the purified GST-TRß completely retained its physiological activity. Our results indicated that the recombinant TRß was secreted extracellularly using the silk fibroin signal peptide sequence. Moreover, we found that the expression system of silkworms was applicable to nuclear proteins.

Cell-Free Protein Synthesis Approach to Biosensing hTRß-Specific Endocrine Disruptors.

Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor ß in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

Loss of tyrosine phosphorylation at Y406 abrogates the tumor suppressor functions of the thyroid hormone receptor ß.

We have recently identified that phosphorylation at tyrosine (Y)406 is critical for the tumor suppressor functions of the thyroid hormone receptor ß1 (TRß) in a breast cancer line. However, still unclear is whether the critical tumor suppressor role of phosphorylated Y406 of TRß is limited to only breast cancer cells or could be extended to other cell types. In the present studies, we addressed this question by stably expressing TRß, a mutated TRß oncogene (PV), or a TRß mutated at Y406 (TRßY406F) in rat PCCL3 thyroid follicular cells and evaluated their tumor characteristics in athymic mice with elevated thyroid stimulating hormone. PCCL3 cells stably expressing PV (PCCL3-PV), TRßY406F (PCCL3-TRßY406F), or vector only (PCCL3-Neo) developed tumors with sizes in the rank order of TRßY406F>PV = Neo, whereas PCCL3 cells expressing TRß (PCCL3-TRß) barely developed tumors. As evidenced by markedly elevated Ki67, cyclin D1, and p-Rb protein abundance, proliferative activity was high in PV and TRßY406F tumors, but low in TRß tumors. These results indicate that TRß acted as a tumor suppressor in PCCL3 cells, whereas TRßY406F and PV had lost tumor suppressor activity. Interestingly, TRßY406F tumors had very low necrotic areas with decreased TNFα-NFκB signaling to lower apoptotic activity. In contrast, PV tumors had prominent large necrotic areas, with no apparent changes in TNFα-NFκB signaling, indicating distinct oncogenic activities of mutant PV and TRßY406F. Thus, the present studies uncovered a novel mechanism by which TRß could function as a tumor suppressor through modulation of the TNFα-NFκB signaling. © 2016 Wiley Periodicals, Inc.

In silico modelling of prostacyclin and other lipid mediators to nuclear receptors reveal novel thyroid hormone receptor antagonist properties.

Prostacyclin (PGI2) is a key mediator involved in cardiovascular homeostasis, acting predominantly on two receptor types; cell surface IP receptor and cytosolic peroxisome proliferator activated receptor (PPAR) ß/δ. Having a very short half-life, direct methods to determine its long term effects on cells is difficult, and little is known of its interactions with nuclear receptors. Here we used computational chemistry methods to investigate the potential for PGI2, beraprost (IP receptor agonist), and GW0742 (PPARß/δ agonist), to bind to nuclear receptors, confirmed with pharmacological methods. In silico screening predicted that PGI2, beraprost, and GW0742 have the potential to bind to different nuclear receptors, in particular thyroid hormone ß receptor (TRß) and thyroid hormone α receptor (TRα). Docking analysis predicts a binding profile to residues thought to have allosteric control on the TR ligand binding site. Luciferase reporter assays confirmed that beraprost and GW0742 display TRß and TRα antagonistic properties; beraprost IC50 6.3 × 10(-5)mol/L and GW0742 IC50 4.9 × 10(-6) mol/L. Changes to triiodothyronine (T3) induced vasodilation of rat mesenteric arteries measured on the wire myograph were measured in the presence of the TR antagonist MLS000389544 (10(-5) mol/L), beraprost (10(-5) mol/L) and GW0742 (10(-5) mol/L); all significantly inhibited T3 induced vasodilation compared to controls. We have shown that both beraprost and GW0742 exhibit TRß and TRα antagonist behaviour, and suggests that PGI2 has the ability to affect the long term function of cells through binding to and inactivating thyroid hormone receptors.

(-) Arctigenin and (+) pinoresinol are antagonists of the human thyroid hormone receptor ß.

Lignans are important biologically active dietary polyphenolic compounds. Consumption of foods that are rich in lignans is associated with positive health effects. Using modeling tools to probe the ligand-binding pockets of molecular receptors, we found that lignans have high docking affinity for the human thyroid hormone receptor ß. Follow-up experimental results show that lignans (-) arctigenin and (+) pinoresinol are antagonists of the human thyroid hormone receptor ß. The modeled complexes show key plausible interactions between the two ligands and important amino acid residues of the receptor.

Ligand-dependent corepressor acts as a novel corepressor of thyroid hormone receptor and represses hepatic lipogenesis in mice.

BACKGROUND & AIMS: Transcriptional co-regulators assist nuclear receptors to control the transcription and maintain the metabolic homeostasis. Ligand-dependent corepressor (LCOR) was reported to function as a transcriptional corepressor in vitro. We found LCOR expression decreased in fatty livers of leptin-deficient (ob/ob) mice, diet-induced obese mice, as well as patients, suggesting LCOR may play a role in lipid homeostasis. We sought to investigate the physiological role of LCOR in vivo and elucidate the underlining molecular mechanisms. METHODS: The effect of LCOR on hepatic lipid accumulation and thyroid hormone receptor (TR) mediated expression of lipogenic genes was studied in vitro and in vivo. RESULTS: Ectopic expression of LCOR via intravenous infection with LCOR adenovirus decreased the hepatic triglyceride level in wild type, ob/ob, and diet-induced obese mice. Interestingly, overexpression of LCOR repressed the thyroid hormone induced expression of lipogenic genes and non-lipogenic genes, and ameliorated hepatic steatosis in obese mice, suggesting that LCOR might regulate lipogenesis as a novel TR corepressor. Furthermore, our study revealed that LCOR could interact with TRß1 in the presence of the ligand, which resulted in competitive binding and reduced recruitment of steroid receptor coactivator-1/3 (SRC-1/3) to the promoter region of TR target genes. CONCLUSIONS: Our data suggest that LCOR is likely to suppress TRß1-mediated hepatic lipogenesis by decreasing binding and recruitment of SRCs to TRß1. Our study reveals the physiological function of hepatic LCOR in lipid metabolism and the mechanism by which LCOR regulates lipogenesis. Hepatic LCOR may be a potential target for treating hepatic steatosis.

A novel p.E311K mutation of thyroid receptor beta gene in resistance to thyroid hormone syndrome, inherited in autosomal recessive trait.

Resistance to thyroid hormone (RTH) syndrome is caused by mutations in THRB gene and is inherited mainly as an autosomal dominant trait with dominant negative effect. Most of up-to-now described RTH cases were heterozygous. We studied a 19-year-old woman presenting severe mental impairment, hyperkinetic behavior, learning disability, hearing loss, tachycardia, goiter, strabismus, nystagmus, and normal stature. The laboratory findings revealed elevated TSH, T3, and T4 serum levels. Her parents were healthy with normal serum level of TSH, fT3, and fT4. Sequence based prediction of a substitution was analyzed by SDM, PolPhen, and SNAP software whereas structural visualizations were performed in UCSF Chimera. We found a novel mutation in THRB gene in position 1216 (G to A transition, codon 311) resulting in novel Glu-311-Lys (p.E311K) substitution, homozygous in proband presenting with severe symptoms of RTH and heterozygous in both of her healthy parents, thus suggesting autosomal recessive mode of inheritance. p.E311K substitution was not found in 50 healthy, unrelated individuals. p.E311K was shown to be deleterious by SDM, PolPhen, and SNAP software. Structural visualizations of mutated protein performed by UCSF Chimera software disclosed a loss of hydrogen bonds between E311, R383, and R429 along with abnormal residue-residue contact between K311 and L377. This is a very rare case of a homozygous mutation in a patient with severe symptoms of RTH and lack of symptoms in both heterozygous parents. Although, computational analyses have provided the evidence that p.E311K substitution may affect THRB function, lack of dominant negative effect typical for THRB mutations could not be explained by structure-based modeling. Further in vitro analysis is required to assess the functional consequences of this substitution.

Study for the binding affinity of thyroid hormone receptors based on machine learning algorithm.

Long-term exposure of exogenous compounds to thyroid hormone receptors (TRs) may lead to thyroid dysfunction. Quantitative structure-activity relationship (QSAR) is expected to predicting the binding affinity of compounds to TR. In this work, two comprehensive and large datasets for TRα and TRß were collected and investigated. Five machine learning models were established to predict the pIC50 of compounds. Meanwhile, the reliability of the models was ensured by a variety of evaluation parameters. The results showed that the support vector regression model exhibited the best robustness and external prediction ability (r2train = 0.77, r2test = 0.78 for TRα, r2train = 0.78, r2test = 0.80 for TRß). We have proposed an appropriate mechanism for explaining the TR binding affinity of a compound. The molecular volume, mass, and aromaticity affected the activity of TRα. Molecular weight, electrical properties and molecular hydrophilicity played a significant role in the binding affinity of compounds to TRß. We also characterized the application domain of the model. Finally, the obtained models were utilized to predict the TR binding affinities of 109 compounds from the list of endocrine disruptors. Therefore, this model is expected to be an effective tool for alerting the effects of exogenous compounds on the thyroid system.

[The cloning, expression and the binding ability with TRß1 of retinoid X receptor-α gene].

Retinoid X receptor-α (RXR-α), a member of nuclear receptor family, is capable of mediating retinoid signaling pathways and plays a critical role in regulating target gene transcription. To further study the function of RXR-α, abundant of recombinant RXR-α protein in hand is necessary. In this study an intact RXR-α coding sequence was amplified by RT-PCR and subsequently inserted into expression plasmid vector pQE-30Xa to form the recombinant construct of pQE-30Xa/RXR-α. Thereafter, competent bacteria Escherichia coli M15 [PREP4] was transformed and the expression of RXR-α was induced by adding IPTG to the medium. Bacterially expressed recombinant RXR-α was purified by Ni-NTA affinity chromatography and verified by SDS-PAGE and Western blotting analyses. The results showed that a protein, with the molecular mass around 50 kDa, could be selectively recognized by anti-RXR-α antibody. Co-immunoprecipitation assay indicated that this recombinant RXR-α could effectively bind TRß1 to form a heterodimer, which could specifically bind the target DNA fragment. This was confirmed by EMSA. In conclusion, the recombinant human retinoid X receptor-α was prepared successfully, which makes a basic for further study of its function.

Negative regulation by thyroid hormone receptor requires an intact coactivator-binding surface.

Thyroid hormone (TH) action is mediated by TH receptors (TRs), which are members of the nuclear hormone receptor superfamily. In vitro studies have demonstrated that TR activity is regulated by interactions with corepressor and coactivator proteins (CoRs and CoAs, respectively). TH stimulation is thought to involve dissociation of CoRs and recruitment of CoAs to the liganded TR. In contrast, negative regulation by TH is thought to occur via recruitment of CoRs to the liganded TR. The physiological role of CoAs bound to TRs, however, has yet to be defined. In this study, we used gene-targeting techniques to mutate the TR-beta locus within its activation function-2 (AF-2) domain (E457A). This mutation was chosen because it completely abolished CoA recruitment in vitro, while preserving normal triiodothyronine (T3) binding and CoR interactions. As expected, TH-stimulated gene expression was reduced in homozygous E457A mice. However, these animals also displayed abnormal regulation of the hypothalamic-pituitary-thyroid axis. Serum thyroxine, T3, and thyroid-stimulating hormone (TSH) levels and pituitary Tshb mRNA levels were inappropriately elevated compared with those of WT animals, and L-T3 treatment failed to suppress serum TSH and pituitary Tshb mRNA levels. Therefore, the AF-2 domain of TR-beta is required for positive and, paradoxically, for negative regulation by TH in vivo.

Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms.

BACKGROUND: Thyroid receptors, TRalpha and TRbeta, are involved in important physiological functions such as metabolism, cholesterol level and heart activities. Whereas metabolism increase and cholesterol level lowering could be achieved by TRbeta isoform activation, TRalpha activation affects heart rates. Therefore, beta-selective thyromimetics have been developed as promising drug-candidates for treatment of obesity and elevated cholesterol level. GC-1 [3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)-phenoxy acetic acid] has ability to lower LDL cholesterol with 600- to 1400-fold more potency and approximately two- to threefold more efficacy than atorvastatin (Lipitor(c)) in studies in rats, mice and monkeys. RESULTS: To investigate GC-1 specificity, we solved crystal structures and performed molecular dynamics simulations of both isoforms complexed with GC-1. Crystal structures reveal that, in TRalpha Arg228 is observed in multiple conformations, an effect triggered by the differences in the interactions between GC-1 and Ser277 or the corresponding asparagine (Asn331) of TRbeta. The corresponding Arg282 of TRbeta is observed in only one single stable conformation, interacting effectively with the ligand. Molecular dynamics support this model: our simulations show that the multiple conformations can be observed for the Arg228 in TRalpha, in which the ligand interacts either strongly with the ligand or with the Ser277 residue. In contrast, a single stable Arg282 conformation is observed for TRbeta, in which it strongly interacts with both GC-1 and the Asn331. CONCLUSION: Our analysis suggests that the key factors for GC-1 selectivity are the presence of an oxyacetic acid ester oxygen and the absence of the amino group relative to T3. These results shed light into the beta-selectivity of GC-1 and may assist the development of new compounds with potential as drug candidates to the treatment of hypercholesterolemia and obesity.

Only subtle protein conformational adaptations are required for ligand binding to thyroid hormone receptors: simulations using a novel multipoint steered molecular dynamics approach.

Thyroid hormone receptors (TR) are hormone-dependent transcription regulators that play a major role in human health, development, and metabolic functions. The thyroid hormone resistance syndrome, diabetes, obesity, and some types of cancer are just a few examples of important diseases that are related to TR malfunctioning, particularly impaired hormone binding. Ligand binding to and dissociation from the receptor ultimately control gene transcription and, thus, detailed knowledge of binding and release mechanisms are fundamental for the comprehension of the receptor's biological function and development of pharmaceuticals. In this work, we present the first computational study of ligand entry into the ligand binding domain (LBD) of a nuclear receptor. We report molecular dynamics simulations of ligand binding to TRs using a generalization of the steered molecular dynamics technique designed to perform single-molecule pulling simulations along arbitrarily nonlinear driving pathways. We show that only gentle protein movements and conformational adaptations are required for ligand entry into the LBDs and that the magnitude of the forces applied to assist ligand binding are of the order of the forces involved in ligand dissociation. Our simulations suggest an alternative view for the mechanisms ligand binding and dissociation of ligands from nuclear receptors in which ligands can simply diffuse through the protein surface to reach proper positioning within the binding pocket. The proposed picture indicates that the large-amplitude protein motions suggested by the apo- and holo-RXRalpha crystallographic structures are not required, reconciling conformational changes of LBDs required for ligand entry with other nuclear receptors apo-structures that resemble the ligand-bound LBDs.

Design and synthesis of novel 3-hydroxy-cyclobut-3-ene-1,2-dione derivatives as thyroid hormone receptor beta (TR-beta) selective ligands.

Design and synthesis of a novel 3-hydroxy-cyclobut-3-ene-1,2-dione derivatives are reported and their in vitro thyroid hormone receptor selectivity has been evaluated in the thyroid luciferase receptor assay. The 3-[3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)-phenylamino]-4-hydroxy-cyclobut-3-ene-1,2-dione 21 has shown selectivity towards thyroid hormone receptor beta.

The Affinity of Brominated Phenolic Compounds for Human and Zebrafish Thyroid Receptor ß: Influence of Chemical Structure.

Brominated phenolic compounds (BPCs) are found in the environment, and in human and wildlife tissues, and some are considered to have endocrine disrupting activities. The goal of this study was to determine how structural differences of 3 BPC classes impact binding affinities for the thyroid receptor beta (TRß) in humans and zebrafish. BPC classes included halogenated bisphenol A derivatives, halogenated oxidative transformation products of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and brominated phenols. Affinities were assessed using recombinant TRß protein in competitive binding assays with 125I-triiodothyronine (125I-T3) as the radioligand. Zebrafish and human TRß displayed similar binding affinities for T3 (Ki = 0.40 and 0.49 nM) and thyroxine (T4, Ki = 6.7 and 6.8 nM). TRß affinity increased with increasing halogen mass and atomic radius for both species, with the iodinated compounds having the highest affinity within their compound classes. Increasing halogen mass and radius increases the molecular weight, volume, and hydrophobicity of a compound, which are all highly correlated with increasing affinity. TRß affinity also increased with the degree of halogenation for both species. Human TRß displayed higher binding affinities for the halogenate bisphenol A compounds, whereas zebrafish TRß displayed higher affinities for 2,4,6-trichlorophenol and 2,4,6-trifluorophenol. Observed species differences may be related to amino acid differences within the ligand binding domains. Overall, structural variations impact TRß affinities in a similar manner, supporting the use of zebrafish as a model for TRß disruption. Further studies are necessary to investigate how the identified structural modifications impact downstream receptor activities and potential in vivo effects.

Thyroid hormone action: a binding contract.

Thyroid hormones are critical for differentiation, growth, and metabolism. A new study investigating the biological role of the TH receptor TR-beta has demonstrated that DNA binding is critical for most of its functions, but also suggests that novel mechanisms independent of DNA binding may contribute to regulation of auditory function by TR-beta.

Structural rearrangements in the thyroid hormone receptor hinge domain and their putative role in the receptor function.

The thyroid hormone receptor (TR) D-domain links the ligand-binding domain (LBD, EF-domain) to the DNA-binding domain (DBD, C-domain), but its structure, and even its existence as a functional unit, are controversial. The D domain is poorly conserved throughout the nuclear receptor family and was originally proposed to comprise an unfolded hinge that facilitates rotation between the LBD and the DBD. Previous TR LBD structures, however, have indicated that the true unstructured region is three to six amino acid residues long and that the D-domain N terminus folds into a short amphipathic alpha-helix (H0) contiguous with the DBD and that the C terminus of the D-domain comprises H1 and H2 of the LBD. Here, we solve structures of TR-LBDs in different crystal forms and show that the N terminus of the TRalpha D-domain can adopt two structures; it can either fold into an amphipathic helix that resembles TRbeta H0 or form an unstructured loop. H0 formation requires contacts with the AF-2 coactivator-binding groove of the neighboring TR LBD, which binds H0 sequences that resemble coactivator LXXLL motifs. Structural analysis of a liganded TR LBD with small angle X-ray scattering (SAXS) suggests that AF-2/H0 interactions mediate dimerization of this protein in solution. We propose that the TR D-domain has the potential to form functionally important extensions of the DBD and LBD or unfold to permit TRs to adapt to different DNA response elements. We also show that mutations of the D domain LXXLL-like motif indeed selectively inhibit TR interactions with an inverted palindromic response element (F2) in vitro and TR activity at this response element in cell-based transfection experiments.

Recent advances in the development of agonists selective for beta1-type thyroid hormone receptor.

This mini-review will provide an overview on the recent design principles and structure-activity-relationship of beta-selective thyroid hormone receptor (TR) agonists. The prospects for the treatment of metabolic diseases as dyslipidemia with TRbeta-selective ligands are considerable enough so as to avoid cardiovascular acceleration mediated through TRalpha.

Recognition and accommodation at the androgen receptor coactivator binding interface.

Prostate cancer is a leading killer of men in the industrialized world. Underlying this disease is the aberrant action of the androgen receptor (AR). AR is distinguished from other nuclear receptors in that after hormone binding, it preferentially responds to a specialized set of coactivators bearing aromatic-rich motifs, while responding poorly to coactivators bearing the leucine-rich "NR box" motifs favored by other nuclear receptors. Under normal conditions, interactions with these AR-specific coactivators through aromatic-rich motifs underlie targeted gene transcription. However, during prostate cancer, abnormal association with such coactivators, as well as with coactivators containing canonical leucine-rich motifs, promotes disease progression. To understand the paradox of this unusual selectivity, we have derived a complete set of peptide motifs that interact with AR using phage display. Binding affinities were measured for a selected set of these peptides and their interactions with AR determined by X-ray crystallography. Structures of AR in complex with FxxLF, LxxLL, FxxLW, WxxLF, WxxVW, FxxFF, and FxxYF motifs reveal a changing surface of the AR coactivator binding interface that permits accommodation of both AR-specific aromatic-rich motifs and canonical leucine-rich motifs. Induced fit provides perfect mating of the motifs representing the known family of AR coactivators and suggests a framework for the design of AR coactivator antagonists.

Toxic effects of triclosan on a zebrafish (Danio rerio) liver cell line, ZFL.

Triclosan (TCS, 5-chloro-2-(2,4-dichlorophenoxy) phenol) is an antimicrobial agent widely used in personal care products. It has been detected in surface water, soil, aquatic species, and even humans. In this study, we used zebrafish (Danio rerio) as a model to test the hypothesis that TCS exhibits toxic effects by interacting with thyroid hormone receptor ß (TRß) and aryl hydrocarbon receptor (AhR) and by inducing the transcription of thyroid hormone (TH)-associated genes and affecting phase I and phase II enzymes. The median lethal concentrations (LC50) of TCS in zebrafish embryos/larvae and a zebrafish liver cell line (ZFL) were first determined. Hatched larvae were most sensitive to TCS exposure, with LC50 values ranging from 1.26 to 1.46µM for 96h after hatching exposure. The major effect of TCS was delayed hatching which occurred from 1.13µM. The constructed GFP-zfTRß fusion protein revealed the subcellular location of zfTRß as the nucleus in both T3-induced and uninduced states, adding to the difficulty of studying TCS action on thyroid hormone receptors in ZFL cells. TCS had neither agonistic nor antagonistic effects on zfTRßLBD or AhR from the reporter gene systems. Ethoxyresorufin-o-deethylase (EROD) assay suggested that TCS is a weak P4501a (Cyp1a) agonist at 5µM and that it inhibits cytochrome Cyp1a activity induced by benzo(a)pyrene (BaP). In time course-based mRNA profiling in ZFL cells, 4-h exposure to TCS caused a significant (up to 37.5-fold) inhibition of Cyp1a at 2.5µM. An overall inhibition of liver phase I and II gene transcription at 4h exposure indicates the possible quick catabolism of TCS. Our findings suggest that TCS is not a TH mimic that affects TH-related gene expression. The impairment of Cyp1a mRNA expression could be due to stimulation by other stressors such as oxidative stress, warranting further investigation into the underlying mechanism in zebrafish.

Editor's Highlight: Structure-Based Investigation on the Binding and Activation of Typical Pesticides With Thyroid Receptor.

A broad range of pesticides have been reported to interfere with the normal function of the thyroid endocrine system. However, the precise mechanism(s) of action has not yet been thoroughly elucidated. In this study, 21 pesticides were assessed for their binding interactions and the potential to disrupt thyroid homeostasis. In the GH3 luciferase reporter gene assays, 5 of the pesticides tested had agonistic effects in the order of procymidone > imidacloprid > mancozeb > fluroxypyr > atrazine. 11 pesticides inhibited luciferase activity of T3 to varying degrees, demonstrating their antagonistic activity. And there are 4 pesticides showed mixed effects when treated with different concentrations. Surface plasmon resonance (SPR) biosensor technique was used to directly measure the binding interactions of these pesticides to the human thyroid hormone receptor (hTR). 13 pesticides were observed to bind directly with TR, with a KD ranging from 4.80E-08 M to 9.44E-07 M. The association and disassociation of the hTR/pesticide complex revealed 2 distinctive binding modes between the agonists and antagonists. At the same time, a different binding mode was displayed by the pesticides showed mix agonist and antagonist activity. In addition, the molecular docking simulation analyses indicated that the interaction energy calculated by CDOCKER for the agonists and antagonists correlated well with the KD values measured by the surface plasmon resonance assay. These results help to explain the differences of the TR activities of these tested pesticides.