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Molecular mechanism of omega-3 fatty acid transport into the brain

omega-3脂肪酸转运至大脑的分子机制

基本信息

批准号：
10307571
负责人：
Filippo Mancia
金额：
$ 24.3万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-12-01 至 2023-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10307571
关键词：
Affinity Binding Biochemical Biological Assay Biophysics Blood Blood - brain barrier anatomy Blood-Retinal Barrier Brain Cells Cellular Assay Collaborations Complement Complex Cryoelectron Microscopy Data Data Collection Detergents Development Developmental Delay Disorders Docosahexaenoic Acids Drug Delivery Systems Endothelial Cells Exhibits Eye Fab Immunoglobulins Family member Fatty Acids Foundations Head Human Image Integral Membrane Protein Intervention Knock-out Ligands Link Lipids Lysophosphatidylcholines Measures Mediating Membrane Methods Microcephaly Modeling Molecular Molecular Conformation Mus N Domain Nature Omega-3 Fatty Acids Patients Physiological Physiology Polyunsaturated Fatty Acids Positioning Attribute Preparation Prodrugs Recombinants Research Resolution Route Salmon Sampling Silver Singapore Structure System Testing Transmembrane Domain Transport Process Water analog base brain cell design experimental study fatty acid transport loss of function mutation member nanodisk novel particle protein transport proteoliposomes rational design reconstitution small molecule structural biology uptake

项目摘要

ABSTRACT Major Facilitator Superfamily Domain containing 2A (MFSD2A) is a 58 kDa integral membrane protein that is highly expressed within endothelial cells of the blood-brain and blood-retinal barriers where it mediates uptake of the ω-3 fatty acid docosahexaenoic acid (DHA) into the brain and eyes respectively. DHA comprises up to 20% of total brain lipids. Despite this, the brain cannot synthesise DHA de novo and MFSD2A-mediated transport is the primary route by which the brain acquires DHA. Mouse MFSD2A KO models exhibit severe brain DHA deficiency and generalized microcephaly, and human patients with homozygous loss-of-function mutations in MFSD2A present with severe microcephaly and developmental delay. Previous cell-based studies have demonstrated that MFSD2A transports DHA in the form of lysophosphatidylcholine (LPCDHA), but not unesterified fatty acid, in a Na+-dependent manner. Similar to all other MFS family members, MFSD2A has twelve transmembrane domains but unlike most MFS proteins that transport water soluble compounds, MFSD2A transports lysolipids, raising the possibility of a unique transport mechanism. It has been hypothesized that MFSD2A transports mono-acyls chain lipids across membranes by flipping the LPC between membrane leaflets, acting essentially as a “flippase”. Understanding MFSD2A- mediated transporter has the potential to aid drug delivery to the brain. Proof of concept studies have indicated that small molecules covalently linked to LPC can be transported by MFSD2A, potentially providing a new platform for drug delivery across the blood brain barrier – a major bottleneck in neurotherapeutic development. Currently, there are no known atomic resolution structures of MFSD2A and a limited understanding of its molecular transport mechanism. This has hindered our understanding of who DHA is delivered into brain and precludes structure-based LPC-prodrug design for neurotherapeutic intervention. Here we present the first structure of MFSD2A, in an apo inward-open conformation - in complex with a Fab to increase the size of and introduce features to the imaged particles – determined using single particle cryo electron microscopy to 3.45 Å resolution. In collaboration with, Dr. David Silver at DUKE-NUS (Singapore), a pioneer in the MSFD2A physiology field, we have developed biochemical assays to probe structure-based functional hypotheses for MSFD2A in cells, solution, and proteoliposomes. We are proposing to determine the structures of MSFD2A in complex with its ligand and in an outward-facing conformation, and to use our newly developed biochemical assays to probe and fully understand the molecular features of these structures. The results generated from this application will elucidate the molecular-mechanism behind MSFD2A-mediated LPCDHA transport and will provide information regarding how DHA enters the brain. Furthermore, this research has the potential to lay the foundation for the rational design of neurotherapeutics that “hijack” MFSD2A for delivery across the blood brain barrier.

抽象的含有 2A (MFSD2A) 的主要促进子超家族结构域是一种 58 kDa 的整合膜蛋白，在血脑和血视网膜屏障的内皮细胞中高度表达，并介导摄取 ω-3脂肪酸二十二碳六烯酸（DHA）分别进入大脑和眼睛。 DHA 包含多达占脑总脂质的20%。尽管如此，大脑无法从头合成 DHA 和 MFSD2A 介导的运输是大脑获取DHA的主要途径。小鼠 MFSD2A KO 模型表现出严重的脑部 DHA 缺陷和全身性小头畸形，以及具有纯合功能丧失突变的人类患者 MFSD2A 表现为严重的小头畸形和发育迟缓。先前的细胞研究表明 MFSD2A 以以下形式转运 DHA：溶血磷脂酰胆碱 (LPCDHA)，但不是未酯化的脂肪酸，以 Na+ 依赖性方式。与所有类似与其他 MFS 家族成员一样，MFSD2A 有 12 个跨膜结构域，但与大多数 MFS 蛋白不同运输水溶性化合物，MFSD2A 运输溶血脂，提高了独特运输的可能性机制。据推测，MFSD2A 通过以下方式跨膜转运单酰基链脂质：在膜小叶之间翻转 LPC，本质上充当“翻转酶”。了解 MFSD2A- 介导的转运蛋白有可能帮助药物输送到大脑。概念验证研究表明与 LPC 共价连接的小分子可以通过 MFSD2A 运输，这可能提供一种新的跨越血脑屏障的药物输送平台——神经治疗发展的主要瓶颈。目前，尚无已知的 MFSD2A 原子分辨率结构，对其的了解也有限。分子运输机制。这阻碍了我们对谁将 DHA 输送到大脑以及排除了基于结构的 LPC 前药设计用于神经治疗干预。在这里，我们展示了 MFSD2A 的第一个结构，呈 apo 向内开放构象 - 与 Fab 复合，增加成像颗粒的尺寸并引入特征——使用单颗粒冷冻确定电子显微镜分辨率为 3.45 Å。与 DUKE-NUS（新加坡）的 David Silver 博士合作，作为 MSFD2A 生理学领域的先驱，我们开发了生化测定法来探测基于结构的 MSFD2A 在细胞、溶液和蛋白脂质体中的功能假设。我们建议确定 MSFD2A 与其配体复合且呈向外构象的结构，并使用我们的新开发了生化测定法来探测并充分了解这些结构的分子特征。这该应用产生的结果将阐明 MSFD2A 介导的分子机制 LPCDHA 运输并提供有关 DHA 如何进入大脑的信息。此外，这项研究有潜力为“劫持”MFSD2A 的神经治疗药物的合理设计奠定基础穿过血脑屏障的输送。