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

OATP是溶质载体转运蛋白（SLC）家族的一员，在40多种动物中介导内源性和外源性分子的钠非依赖性摄取。OATP 2A 1，也称为胰头素转运蛋白（PGT），是一种广泛表达的OATP，负责胰头素生物活性脂质的细胞摄入（流入）和分泌（流出）。PGT通过调节配体与配体的细胞外可及性，在三兰素的生理功能中发挥重要作用。一些OATP已被证明使用调节其细胞功能的保守结构基序寡聚化。然而，由于缺乏一个三维结构和技术挑战，探针膜蛋白寡聚化，结构功能机制的OATP仍然很差的特点。该研究计划的总体长期目标是了解OATP细胞功能的结构机制，短期重点是研究PGT作为原型OATP。更具体地说，我们建议建立的方法论的理由，将导致一个高分辨率的X-射线晶体结构的PGT的测定，并调查的作用，OATPs的寡聚化PGT介导的前列腺素细胞转运。 本课题旨在从原子水平上阐明PGT转运拟南芥素的分子机制。使用分子模型作为指导，将开发PGT过表达，纯化和优化程序，使结构生物学研究的转运蛋白使用X射线晶体学。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获得在原子水平上的溶质运输的理解OATP。在上述开发的工具的帮助下，我们的目标是获得衍射晶体，这将使我们能够解决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II旨在进一步表征OATP寡聚化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OATP的寡聚化图谱，并探索寡聚化在PGT转运功能中的调节作用。 除了提供更好的多尺度理解的millandins调控，这个程序对OATPs将有很大的影响，在许多研究领域以外的结构生物学，如细胞生物学，生态学和兽医科学。来自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细胞测定，X射线晶体学，同步加速器实验和药理学方面发展强大的专业知识。这种培训目前需求量很大，因此受训人员将在生命科学领域担任急需的合格职位。

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.