Structural basis of receptor-mediated cellular vitamin A uptake

摘要 维生素A是所有哺乳动物的必需营养素。许多生物过程，包括最重要的视觉过程，是 至关重要的是，它依赖于充足的供应才能正常发挥作用。维生素A代谢的改变会导致 广泛的眼部缺陷并导致失明。视黄醇(维生素A酒精)是主要的血液循环 在禁食状态下形成维生素A。在需要的时候(即在没有摄入维生素A的情况下)，以便 将维生素A分配到靶外周组织，视黄醇是从肝脏的血液中释放出来的 体内维生素的储存部位，与视黄醇结合蛋白(RBP)结合。在细胞内，视黄醇结合特定的 细胞内载体，即细胞视黄醇结合蛋白，它是活性维生素的前体 A型：视黄醛和维甲酸，维甲酸是调节特定核受体的配体 数百个目标基因的转录。视黄醇-RBP复合体如何释放视黄醇 被细胞内化几十年来一直是争论的主题。STRA6--可能的质膜受体 对于RBP，于2007年确定。 我们首次通过单粒子冷冻测定了在两面体中重组的Danio rerio的STRA6的结构。 电子显微镜的分辨率为3.9？我们的结构揭示了视黄醇转变为视黄醇的可能机制 以STRA6介导的方式从RBP跨膜。它还显示出意想不到的关联 STRA6与钙调蛋白结合，提出视黄醇代谢与钙相关的假说 动态平衡，我们在生物物理和细胞背景下进行了研究。在这里，我们建议理解如何 该系统在分子水平上以机械方式工作。特别是，我们的目标是调查RBP和STRA6是如何 相互作用以调节视黄醇交换，膜在这一过程中所起的作用，以及整个过程是如何 受不同钙水平的影响。为此，我们计划采取综合方法，包括结构上的 生物学、分子动力学(MD)模拟、生物物理实验和视黄醇摄取分析。我们会 切换到哺乳动物系统，以便更好地将我们的结果与基于细胞的观察相关联，并执行 在接近自然环境下的脂质填充纳米盘的结构实验。 为了支持所提出的实验，我们提供了关于哺乳动物STRA6结构的初步数据 在载脂蛋白形式和与RBP的络合物中，关于侧重于分子的MD模拟-详细的 视黄醇从RBP释放到STRA6和从STRA6释放到膜的机械方面，以及 我们将使用的所有生物物理和生化分析都已到位的证据。多方面的、协同的 我们建议在此应用程序中进行的机械研究将使基于结构的详细理解成为可能 STRA6介导的视黄醇摄取。这是一个极其重要的过程，特别是在诸如 眼睛，它依赖于严格控制视黄醇水平来实现适当的生理功能。

ABSTRACT Vitamin A is an essential nutrient for all mammals. Many biological processes, including and foremost vision, are crucially dependent on its adequate supply for proper function. Alterations of vitamin A metabolism can result in a wide spectrum of ocular defects and lead to blindness. Retinol (vitamin A alcohol) is the predominant circulating vitamin A form in the fasting state. In times of need (i.e. in the absence of dietary vitamin A intake), in order to distribute vitamin A to the target peripheral tissues, retinol is released in the bloodstream from the liver, the main body storage site of the vitamin, bound to retinol-binding protein (RBP). Inside the cells, retinol binds specific intracellular carriers, namely cellular retinol-binding proteins, and it serves as a precursor for the active vitamin A forms: retinaldehyde, critical for vision, and retinoic acid, the ligand for specific nuclear receptors that regulate the transcription of hundreds of target genes. How retinol is released from the retinol-RBP complex and internalized by the cell has been subject of debate for decades. STRA6, the putative plasma membrane receptor for RBP, was identified in 2007. We first determined the structure of STRA6, from Danio rerio reconstituted in amphipol, by single-particle cryo- electron microscopy to 3.9 Å resolution. Our structure revealed a possible mechanism for retinol to transition from RBP across the membrane, in a STRA6-mediated manner. It also showed an unexpected association of STRA6 with calmodulin, setting forth the hypothesis of a correlation between retinol metabolism and calcium homeostasis, which we investigated in a biophysical and cellular context. Here we propose to understand how the system works mechanistically at a molecular level. In particular, we aim to investigate how RBP and STRA6 interact to mediate retinol exchange, the role the membrane plays in the process, and how the entire process is impacted by varying calcium levels. To do so, we plan to adopt an integrated approach comprising structural biology, molecular dynamics (MD) simulations, biophysical experiments and retinol-uptake assays. We will switch to a mammalian system so as to better correlate our results with cell-based observations, and to perform structural experiments in the close to native environment of a lipid-filled nanodisc. In support of the proposed experiments, we present preliminary data on the structure of a mammalian STRA6 both in the apo form and in complex with RBP, on the MD simulations focusing on the molecular-detailed mechanistic aspects of the release of retinol from RBP into STRA6, and from STRA6 into the membrane, and evidence that all the biophysical and biochemical assays we will utilize are in place. The multifaceted, synergistic mechanistic studies we propose to carry out in this application will enable detailed structure-based understanding of STRA6-mediated retinol uptake. This is a process of extreme importance, in particular in organs such as the eye, that rely on tight control of retinol levels for proper physiological function.