Analysis of the roles of mutations in thyroid hormone receptor-β by a bacterial biosensor system

Abstract

Mutations in thyroid hormone receptors (TRs) often lead to metabolic and developmental disorders, but patients with these mutations are difficult to treat with existing thyromimetic drugs. In this study, we analyzed six clinically observed mutations in the ligand-binding domain of the human TRb using an engineered bacterial hormone biosensor. Six agonist compounds, including triiodothyronine (T 3 ), thyroxine (T 4 ), 3,5,3 0 -triiodothyroacetic acid (Triac), GC-1, KB-141, and CO-23, and the antagonist NH-3 were examined for their ability to bind to each of the TRb mutants. The results indicate that some mutations lead to the loss of ability to bind to native ligands, ranging from several fold to several hundred fold, while other mutations completely abolish the ability to bind to any ligand. Notably, the effect of each ligand on each TRb mutant in this bacterial system is highly dependent on both the mutation and the ligand; some ligands were bound well by a wide variety of mutants, while other ligands lost their affinity for all but the WT receptor. This study demonstrates the ability of our bacterial system to differentiate agonist compounds from antagonist compounds and shows that one of the TRb mutations leads to an unexpected increase in antagonist ability relative to other mutations. These results indicate that this bacterial sensor can be used to rapidly determine ligand-binding ability and character for clinically relevant TRb mutants. Key Words" thyroid hormone receptor " ligand-binding assay " hormone receptor mutations " engineered biosensor

Maternal Hypothyroxinemia-Induced Neurodevelopmental Impairments in the Progeny

“…TRs play vital roles in normal neurodevelopment as shown by a mutation of the TRβ gene being associated with resistance to thyroid hormone and leading to attention deficit/hyperactivity disorders [44]. The deleterious effects of mutations in TRs also include thyroid diseases, endocrine maladies, and even thyroid tumors [81,82].…”

Cell-Free Protein Synthesis Approach to Biosensing hTRβ-Specific Endocrine Disruptors

“…An inserted mini-intein splicing domain has also been included to stabilize the fold of the NHR and provide a conduit for the ligand-binding signal to the reporter protein. A critical aspect of this multidomain biosensor protein is that it is modular, potentially allowing new biosensors based on alternate human and animal NHRs to be generated by swapping NHR ligand binding domains embedded in the intein domain scaffold. − However, the system still relies on bacterial growth phenotypes for activity quantification and thus requires a minimum overnight incubation to produce a sufficient signal. Also, this assay and other cell-based assays (i.e., bacterial, yeast, and mammalian) for detecting NHR-binding ligands can be affected by the presence of cytotoxic chemicals in samples and poor cellular uptake rates.…”