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）运输。关于LAT2的最低知识，例如特异性和在体内的不明确作用。这促使我们在第一个融资期间深入了解LAT2在TH摄入中的结构功能关系。我们不仅肯定了以前关于LAT2在TH吸收中普遍参与的发现，就像我们在Xenopus laevis卵母细胞中所显示的重要发现，即LAT2主要运输3,3`-T2，而T3却不少，但RT3和RT3也不是T4。 LAT2的向外开放分子模型结合了模型引导的LAT2变化和Th衍生物的摄取数据，因此可以成功地阐明LAT2中的决定因素的决定因素，例如i）i）细胞外3,3`-T2识别位点，II）ii a in Intrate try try in II ii a a inii a a inii a a inii a a inii a a inii a a ini ii a a inii a a a inii a a a inii a a a inii a a a inii a a a ii a ii ii a a ii ii a ii ii ii ii ii ii ii ii a）从这些数据中，我们假设LAT2的额外和细胞内侧的不同识别机制。在初步尝试中，我们实际上观察到亮氨酸排出，但在卵母细胞中没有LAT2的T2导出。 COS-1细胞最近有一些新的初始提示，即T2，T1由LAT3/4导出。然而，出口分子细节尚不清楚。因此，探索LAT家族中TH的不同分子外排机制，我们将调查和比较由LAT2，LAT3或LAT4介导的TH导出关系的结构功能关系。详细介绍，我们将i）生成向外和向内的开放模型，以在细胞内方面推导适当的底物识别模式，ii）在Xenopus laevis卵母细胞中的底物外排测量，用于野生类型和模型引导的LAT突变体，iii，III，III）可能会触发cliribiver the的cllifient and the and lat2 and lat1 and lat1 and lat1或lat 2的转换效应。或在柜台侧的氨基酸。为了了解第三口和底物选择性中LAT1/2和LAT3/4之间的功能差异。专注于T2作为非分类的TH有助于阐明其作用被认为是一种替代性TR配体，或者可能对能源代谢的调节进行调节。我们的项目跨越了生物信息知识分子生物学生物学研究，并有助于未来开发新的新型策略，从而跨越了结构，功能和生理方法。针对其他应用合作伙伴的项目，用于甲状腺细胞和星形胶质细胞等不同细胞（例如甲状腺细胞和星形胶质细胞）中的LAT2，LAT3和LAT4的生理TH外排研究正在补充我们的研究。此外，LAT中T2摄取和T2摄取的决定因素和分子机制的详细知识为未来的药理学干预措施提供了预防和靶向依赖性疾病的药理干预措施。