Searching for transport proteins for TRIAC or DITPA acting as T3/TH substitutes.

点击翻译按钮获取中文摘要

Restriction of Thyroid Hormone (TH)-availability during brain development leads to a severe mental retardation, the Allan-Herndon-Dudley syndrome (AHDS). TH are essential for growth, metabolism and development, especially for the central nervous system. TH and their analogues require transmembrane transporters to mediate their translocation across the cell membranes towards their target the thyroid hormone receptor (TR) located in the cell nucleus. Among known thyroid hormone transmembrane transporters (THTT), the monocarboxylate transporter 8 (MCT8) is the only known THTT that is specific for TH. Several pathogenic mutations in human MCT8 have been identified to be responsible for deficiency of the thyroid hormone T3 in AHDS. In contrast to the thyroid hormones T3 and T4, the TH-analogues DITPA (3,5-diiodothyropropionic acid) and TRIAC (3,5,3'-triiodothyroacetic acid) are not transported by MCT8. Moreover, for TRIAC and DITPA it was recently demonstrated that TR activation occurs also under MCT8 deficiency by circumventing the disturbed TH transporter. Thus both TH-analogues have a clinical potential for the pharmacological interference of TH related disease like AHDS. However, the underlying molecular mechanisms and transport proteins for DITPA and TRIAC are unknown.In preliminary studies we could demonstrate a temperature dependent uptake and concentration dependent saturation of uptake of TRIAC and DITPA, indicating protein mediated translocation.Our proposal is aimed at identifying the transmembrane protein(s) responsible for the uptake of DITPA and/or TRIAC and at characterizing the substrate spectra and transport mechanism, as well as identification of intracellular TH-binding proteins. We will utilize RNA interference (RNAi) screening technology to identify transporter protein(s) for these TH-analogues with a human whole genome library of small interfering RNAs (siRNA). siRNA induced silencing of transporters relevant for TH-analogues will prevent their entrance into cells and thus cause a lack of TR activation. Utilizing a reporter plasmid/gene for TR-activation, we can link a relevant silenced gene to an absent signal in the assay. A knockdown of intracellular TH-binding proteins however, will lead to an increased signal in the assay. Preliminary knockdown studies of MCT8 in HepG2-cells show a clear reduction of TR-activation by T3 and provide a proof of principle for our RNAi screen.In a two-stage strategy, we first perform a whole genome RNAi screen followed by a second validation screen to exclude false positive results. After identification of the respective transporter proteins we will characterize and delineate their determinants for the uptake of TH-analogues. Detailed knowledge about determinants for molecular specificity provides a base on the one hand for optimizing TH-analogues to achieve most efficient functionality and on the other hand for future pharmacological interventions to prevent and target TH dependent diseases.