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

寻找 TRIAC 或 DITPA 的转运蛋白作为 T3/TH 替代品。

基本信息

批准号：
360579008
负责人：
Dr. Gerd Krause
金额：
--
依托单位：
Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP)
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2017
资助国家：
德国
起止时间：
2016-12-31 至 2021-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/360579008?language=en
关键词：
Searching transport proteins TRIAC DITPA

项目摘要

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.

大脑发育过程中甲状腺激素（TH）的可用性受到限制会导致严重的智力障碍，即艾伦-赫恩登-达德利综合征（AHDS）。 TH 对于生长、新陈代谢和发育至关重要，尤其是中枢神经系统。 TH 及其类似物需要跨膜转运蛋白介导其跨细胞膜易位至位于细胞核中的甲状腺激素受体 (TR) 目标。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 (MCT8) 是唯一已知的 TH 特异性 THTT。人类 MCT8 的几种致病性突变已被确定与 AHDS 中甲状腺激素 T3 缺乏有关。与甲状腺激素 T3 和 T4 相比，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）不由 MCT8 转运。此外，最近证明，对于 TRIAC 和 DITPA，TR 激活也可以通过绕过受干扰的 TH 转运蛋白而在 MCT8 缺乏的情况下发生。因此，这两种 TH 类似物都具有对 TH 相关疾病（如 AHDS）进行药理干扰的临床潜力。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 的摄取饱和度依赖于温度和浓度，表明蛋白质介导的易位。我们的建议旨在鉴定负责摄取 DITPA 和/或 TRIAC 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，通过人类小干扰 RNA (siRNA) 全基因组文库来鉴定这些 TH 类似物的转运蛋白。 siRNA 诱导的与 TH 类似物相关的转运蛋白的沉默将阻止它们进入细胞，从而导致 TR 激活的缺乏。利用报告质粒/基因进行 TR 激活，我们可以将相关的沉默基因与检测中缺失的信号联系起来。然而，细胞内 TH 结合蛋白的敲低将导致检测中信号增强。 HepG2 细胞中 MCT8 的初步敲低研究表明 T3 明显降低了 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。在鉴定出各自的转运蛋白后，我们将表征并描述它们摄取 TH-类似物的决定因素。关于分子特异性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针对 TH 依赖性疾病的药理干预提供了基础。