Molecular determinants of different thyroid hormone uptake/ efflux mechanisms for L-type amino acid transporter subtypes

L型氨基酸转运蛋白亚型不同甲状腺激素摄取/流出机制的分子决定因素

• 批准号: 221171143
• 负责人: Dr. Gerd Krause
• 金额: --
• 依托单位: Arbeitsgruppe Structural Bioinformatics and Protein Design
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/221171143?language=en
• 关键词: Molecular; determinants; different; thyroid; hormone

Thyroid hormones (TH) and their derivatives are transported across the cell membrane, amongst other TH transmembrane transporter proteins (THTT), also by the L-type amino acid transporters (LAT). Least was known about Lat2 such as TH specificity and the unclear role in vivo. This motivated us in the first funding period to gain insight into structure-function relationships of Lat2 in TH uptake. We not only affirmed previous findings of a general involvement of Lat2 in the uptake of TH, as important new finding we showed in Xenopus laevis oocytes that Lat2 is mainly transporting 3,3`-T2 and somewhat less T3 but neither rT3 nor T4. Outward-open molecular models of Lat2 combined with uptake data of model-guided Lat2-variants and TH-derivatives enabled a successful elucidation of determinants for TH uptake in Lat2 such as i) the extracellular 3,3`-T2 recognition site, ii) an asymmetrically shaped channel for substrate traversing in the center of Lat2 and iii) a potentially switch site. From these data we hypothesize different recognition mechanisms at the extra and intracellular side of Lat2. In preliminary attempts we in fact observed leucine efflux but no T2 export by Lat2 in oocytes. There are recent new initial hints from COS-1 cells that T2, T1 are exported by LAT3/4. However molecular details for export are unknown.Thus exploring the different molecular efflux mechanisms of TH in the LAT family, we will survey and compare the structure-function relationships of TH export mediated by LAT2, LAT3 or LAT4. In detail we will i) generate outward and inward open models deducing appropriate substrate recognition patterns at the intracellular sides, ii) substrate efflux measurements in Xenopus laevis oocytes, for wild type and model-guided LAT mutants, iii) identification of determinants potentially triggering a switch or changeover function at the intracellular side of LAT2 and LAT3 or LAT4 and iv) clarify transport dependence of TH or amino acids at the counter-side. In order to understand the functional differences between LAT1/2 and LAT3/4 in TH export and substrate selectivity. Focusing on T2 as nonclassical TH contributes to elucidate its role assumed to be an alternative TR ligand or might mediate TH regulation on the energy metabolism.Our project straddles structural, functional and physiological approaches by combining bioinformatics molecular biological and in vitro studies and has the potential to contribute to the future development of new therapeutic strategies. Physiological TH efflux studies for LAT2, LAT3 and LAT4 in diverse cells such as thyrocytes and astrocytes by other applying projects of cooperating partners are complementing our studies. Moreover, detailed knowledge about determinants and molecular mechanisms for both uptake and efflux of T2 in LAT provides the basis for future pharmacological interventions of malfunctions to prevent and target TH dependent diseases.

甲状腺激素（TH）及其衍生物在其他TH跨膜转运蛋白（THTT）中也通过L型氨基酸转运蛋白（LAT）跨细胞膜转运。目前对Lat 2的了解较少，如TH特异性和在体内的作用不清楚。这促使我们在第一个资助期深入了解Lat 2在TH摄取中的结构-功能关系。我们不仅证实了以前的研究结果，一般参与的Lat 2在TH的摄取，作为重要的新发现，我们表明，在非洲爪蟾卵母细胞Lat 2主要是运输3，3 `-T2和T3有点少，但既不是rT 3也不是T4。Lat 2的向外开放的分子模型与模型引导的Lat 2变体和TH衍生物的摄取数据相结合，使得能够成功阐明Lat 2中TH摄取的决定因素，例如i）细胞外3，3 '-T2识别位点，ii）Lat 2中心底物穿过的不对称形状通道和iii）潜在的开关位点。从这些数据中，我们假设不同的识别机制在额外的和细胞内侧的Lat 2。在初步尝试中，我们实际上观察到亮氨酸流出，但没有T2出口的Lat 2在卵母细胞。最近有来自COS-1细胞的新的初步提示，T2，T1是由LAT 3/4导出的。因此，我们将对LAT家族中不同的TH外排机制进行研究，并对LAT 2、LAT 3和LAT 4介导的TH外排的结构与功能关系进行比较。详细地说，我们将i）产生向外和向内的开放模型，推导出细胞内侧的适当底物识别模式，ii）对于野生型和模型指导的LAT突变体，在非洲爪蟾卵母细胞中的底物流出测量，iii）鉴定潜在地触发LAT 2和LAT 3或LAT 4的细胞内侧的转换或转换功能的决定簇，以及阐明TH或氨基酸在对侧的转运依赖性。为了了解LAT 1/2和LAT 3/4在TH输出和底物选择性方面的功能差异。T2是一种非经典的TH，它可能是一种替代性的TR配体，也可能是TH调节能量代谢的重要途径，本研究将生物信息学、分子生物学和体外研究相结合，从结构、功能和生理学的角度对T2进行研究，有望为今后开发新的治疗策略做出贡献。通过合作伙伴的其他应用项目对不同细胞如甲状腺细胞和星形胶质细胞中的LAT 2、LAT 3和LAT 4的生理TH流出研究是对我们的研究的补充。此外，详细了解的决定因素和分子机制的摄取和流出的T2在LAT功能障碍，以预防和目标TH依赖性疾病的未来药理学干预提供了基础。