A multiscale structural understanding of organic anion transporting polypeptide transporters (OATPs) functions

对有机阴离子转运多肽转运蛋白 (OATP) 功能的多尺度结构理解

• 批准号: RGPIN-2021-03486
• 负责人: Audet, Martin
• 金额: $ 2.19万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=741778
• 关键词: multiscale; structural; understanding; organic; anion

OATPs are a family of Solute Carrier Transporters (SLC) that mediates sodium independent uptake of endogenous and xenobiotic molecules in more than 40 animal species. OATP2A1, also called prostaglandins transporter (PGT), is an ubiquitously expressed OATP that is responsible for the cellular intake (influx) and secretion (efflux) of prostaglandins bioactive lipids. PGT plays a central role in prostaglandins physiological functions by regulating prostaglandins receptors extracellular accessibility to their ligands. Some OATPs have been shown to oligomerize using conserved structural motifs that regulate their cellular functions. However, because of the absence of a tridimensional structure and the technical challenge to probe membrane proteins oligomerization, the structure-function mechanism of OATPs remains poorly characterized. The overarching long-term goal of this research program is to understand the structural mechanism of OATPs cellular functions, with short-term focus to study PGT as a prototypical OATP. More specifically, we propose to establish the methodological grounds that will lead to the determination of a high-resolution X-ray crystal structure of PGT, and to investigate the role of OATPs oligomerization on PGT-mediated prostaglandin cellular transport. Theme I aim at defining the molecular mechanism of prostaglandins transport by PGT at an atomic level. Using a molecular model as a guide, will develop PGT overexpression, purification, and optimization procedures, to enable the structural biology study of the transporter using X-ray crystallography. The model will also serve to identify important structural features in PGT regulating the transport function. The results obtained will be used to derive an understanding of solute transport by OATPs at an atomic level. With the help of the tools developed above, we aim at obtaining diffracting crystals that will allow us to solve the high-resolution structure of PGT and launch a larger scale structural biology program on OATPs. Theme II aim at further characterizing the role of OATP oligomerization on their cellular functions. We will use Bioluminescence Resonance Energy Transfer (BRET)-based approaches to determine the oligomerization map of human OATPs in cells and probe the regulatory role of oligomerization in PGT transport functions. In addition to provide a better multiscale understanding of prostaglandins regulation, this program on OATPs will have a high impact in many research areas beyond structural biology such as cell biology, ecology, and veterinary sciences. Students and postdoctoral fellows from my laboratory will develop strong expertise in protein engineering, expression and purification, molecular modelling, BRET cellular assays, X-ray crystallography, synchrotron experimentation, and pharmacology. Such training is currently in high demand, and trainees will therefore fulfill highly needed qualified positions in life sciences.

OATPs是一个溶质载体转运体(SLC)家族，在40多种动物中介导内源和外源分子对钠的非依赖性摄取。OATP2A1，又称前列腺素转运体(PGT)，是一种广泛表达的OATP，负责细胞内前列腺素生物活性脂的摄取和分泌。前列腺素T通过调节前列腺素受体对其配体的胞外可及性，在前列腺素的生理功能中发挥核心作用。一些OATP已被证明使用保守的结构基序来调节其细胞功能而进行寡聚。然而，由于缺乏三维结构和膜蛋白齐聚反应的技术挑战，OATPs的结构-功能机制仍然缺乏研究。这项研究计划的长期目标是了解OATPs细胞功能的结构机制，短期重点是将PGT作为OATP的原型进行研究。更具体地说，我们建议建立导致高分辨率X射线晶体结构确定的方法学基础，并研究OATPS寡聚在PGT介导的前列腺素细胞转运中的作用。主题I旨在从原子水平确定前列腺素转运的分子机制。以分子模型为指导，将开发PGT的过表达、纯化和优化程序，使利用X射线结晶学进行转运蛋白的结构生物学研究成为可能。该模型还将用于确定PGT调节运输功能的重要结构特征。所获得的结果将被用来在原子水平上理解OATP的溶质迁移。借助上面开发的工具，我们的目标是获得衍射晶体，使我们能够解决PGT的高分辨率结构，并在OATP上启动更大规模的结构生物学计划。主题二旨在进一步表征OATP齐聚对其细胞功能的作用。我们将使用基于生物发光共振能量转移(BRET)的方法来确定细胞中人OATPs的寡聚化图谱，并探索寡聚化在PGT运输功能中的调节作用。除了提供对前列腺素调控的更好的多尺度了解外，这个关于OATP的项目将在结构生物学以外的许多研究领域产生高度影响，如细胞生物学、生态学和兽医学。我实验室的学生和博士后研究员将在蛋白质工程、表达和纯化、分子建模、Bret细胞分析、X射线结晶学、同步加速器实验和药理学方面培养强大的专业知识。目前这种培训的需求量很大，因此受训人员将担任生命科学领域急需的合格职位。