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

大脑发育过程中甲状腺激素（TH）的可用性受到限制，导致严重的智力迟钝，即艾伦-赫恩登-达德利综合征（AHDS）。TH是生长、代谢和发育所必需的，特别是对中枢神经系统。TH及其类似物需要跨膜转运蛋白介导其跨细胞膜转运至位于细胞核中的靶甲状腺激素受体（TR）。在已知的甲状腺激素跨膜转运蛋白（THTT）中，单羧酸转运蛋白8（MCT 8）是唯一已知的特异于TH的THTT。已确定人MCT 8中的几种致病性突变是AHDS中甲状腺激素T3缺乏的原因。与甲状腺激素T3和T4相反，TH类似物DITPA（3，5-二碘甲状腺丙酸）和TRIAC（3，5，3 '-三碘甲状腺乙酸）不被MCT 8转运。此外，对于TRIAC和DITPA，最近证明了在MCT 8缺陷下也通过绕过受干扰的TH转运蛋白而发生TR激活。因此，这两种TH类似物具有药理学干扰TH相关疾病如AHDS的临床潜力。然而，DITPA和TRIAC的潜在分子机制和转运蛋白尚不清楚。在初步研究中，我们可以证明TRIAC和DITPA的摄取具有温度依赖性和浓度依赖性饱和，表明蛋白质介导的转运。我们的建议旨在鉴定负责DITPA和/或TRIAC摄取的跨膜蛋白，并表征底物光谱和转运机制，以及细胞内TH结合蛋白的鉴定。我们将利用RNA干扰（RNAi）筛选技术，以确定这些TH-类似物的转运蛋白（S）与人类全基因组文库的小干扰RNA（siRNA）。siRNA诱导的TH-类似物相关转运蛋白的沉默将阻止它们进入细胞，从而导致TR活化的缺乏。利用用于TR激活的报告质粒/基因，我们可以将相关沉默基因与测定中不存在的信号连接。然而，细胞内TH结合蛋白的敲低将导致测定中的信号增加。在HepG 2-细胞中对MCT 8的初步敲低研究显示T3明显降低TR激活，并为我们的RNAi筛选提供了原理证明。在两阶段策略中，我们首先进行全基因组RNAi筛选，然后进行第二次验证筛选以排除假阳性结果。在识别各自的转运蛋白后，我们将表征和描绘其决定因素TH-类似物的摄取。关于分子特异性的决定因素的详细知识一方面为优化TH类似物以实现最有效的功能提供了基础，另一方面为未来预防和靶向TH依赖性疾病的药理学干预提供了基础。

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.