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Structural basis of receptor-mediated cellular vitamin A uptake

受体介导的细胞维生素 A 摄取的结构基础

基本信息

批准号：
10682067
负责人：
George Khelashvili
金额：
$ 65.99万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-04-01 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10682067
关键词：
11 cis Retinal Adopted Affect All-Trans-Retinol Anophthalmos Binding Binding Sites Biochemical Biological Assay Biological Process Biophysics Blindness Blood Circulation Calcium Calmodulin Cell membrane Cells Cellular Retinol Binding Protein Cholesterol Complex Cryoelectron Microscopy Data Data Analyses Defect Development Diffuse Diffusion Disease Dissociation Environment Event Exhibits Eye Fasting Free Energy Genes Genetic Transcription Health Homeostasis Human Hydrophobicity Information Theory Intake Kinetics Knockout Mice Lateral Ligands Link Lipids Liver Mammals Maps Mediating Membrane Metabolism Methods Microphthalmos Modeling Molecular Molecular Conformation Molecular Machines Mutation Names Nuclear Receptors Nutrient Organ Pathologic Pathology Peripheral Phenotype Physiological Play Positioning Attribute Process RBP4 gene Resolution Rest Retinal Pigments Retinaldehyde Retinoids Retinol Binding Proteins Retinol Metabolism Pathway Roentgen Rays Role Sampling Sheep Site Statistical Mechanics Structure Syndrome System Testing Thermodynamics Tissues Tretinoin Vision Visual System Vitamin A Vitamins Wood material Work Zebrafish density design dietary experimental study insight machine learning method malformation molecular dynamics mutant nanodisk new therapeutic target particle protein complex receptor reconstitution retinol binding protein receptor simulation structural biology therapy development uptake visual cycle

项目摘要

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.

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，这主要取决于其充足的供应以实现正常功能。维生素A代谢的改变可导致广泛的眼部缺陷并导致失明。视黄醇（维生素A醇）是主要的循环维生素A在禁食状态下的形式。在需要时（即缺乏膳食维生素A摄入），将维生素A分配到目标外周组织，视黄醇在血液中从肝脏释放，主要是维生素的身体储存部位，与视黄醇结合蛋白（RBP）结合。在细胞内，细胞内载体，即细胞视黄醇结合蛋白，它作为活性维生素的前体 A型：视黄醛，对视力至关重要，视黄酸，调节细胞核内特定受体的配体。数百个靶基因的转录。如何从视黄醇-RBP复合物中释放视黄醇，几十年来一直是争论的主题。STRA 6，推定的质膜受体 RBP是在2007年发现的。我们首先通过单粒子低温冷冻法确定了来自Danio rerio的STRA 6的结构。电子显微镜至3.9 μ m分辨率。我们的结构揭示了一个可能的机制，视黄醇过渡从RBP跨膜，在STRA 6介导的方式。它还显示了一种意想不到的关联， STRA 6与钙调蛋白的关系，提出了视黄醇代谢与钙调蛋白之间相关性的假设。稳态，我们在生物物理和细胞背景下研究。在这里，我们建议了解如何该系统在分子水平上机械地工作。特别是，我们的目标是调查如何RBP和STRA 6 相互作用以介导视黄醇交换，膜在该过程中所起的作用，以及整个过程是如何进行的。受到不同钙水平的影响。为此，我们计划采取一个综合方法，生物学、分子动力学（MD）模拟、生物物理实验和视黄醇摄取测定。我们将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，在接近天然环境的脂质填充纳米盘的结构实验。为了支持所提出的实验，我们提出了哺乳动物STRA 6结构的初步数据，无论是在载脂蛋白的形式和复杂的RBP，在MD模拟侧重于分子详细视黄醇从RBP释放到STRA 6中以及从STRA 6释放到膜中的机制方面，以及我们将使用的所有生物物理和生物化学测定都已到位的证据。多方面的、协同的我们建议在此应用中进行的机理研究将使详细的基于结构的理解成为可能 STRA 6介导的视黄醇摄取。这是一个极其重要的进程，特别是在联合国等机构中，眼睛，依赖于严格控制视黄醇水平以实现适当的生理功能。