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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.

摘要主要促进剂超家族结构域包含2A(MFSD2A)，是一个58 kDa的完整膜蛋白，它是在血-脑内皮细胞和血-视网膜屏障中高表达，在那里它介导摄取 ω-3脂肪酸二十二碳六烯酸(DHA)分别进入大脑和眼睛。DHA最多由占脑脂总量的20%。尽管如此，大脑不能合成DHA和MFSD2A介导的运输是大脑获得DHA的主要途径。小鼠MFSD2AKO模型显示严重的脑DHA 缺乏性和全身性小头畸形和人类纯合子功能丧失突变患者 MFSD2A表现为严重的小头畸形和发育迟缓。以前的基于细胞的研究表明，MFSD2A以以下形式转运DHA 溶血磷脂酰胆碱(LPCDHA)，但不是未酯化脂肪酸，依赖于Na+。与所有类似其他MFS家族成员，MFSD2A有12个跨膜结构域，但不同于大多数MFS蛋白运输水溶性化合物，MFSD2A运输溶质脂，增加了一种独特运输的可能性机制。据推测，MFSD2A通过以下途径跨膜运输单酰基链脂在膜小叶之间翻转LPC，本质上起到“翻转酶”的作用。了解MFSD2A- 介导型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由MFSD2A运输，潜在地提供了一种新的跨血脑屏障给药的平台--这是神经治疗发展的主要瓶颈。目前还没有已知的MFSD2A的原子分辨结构，对其了解也有限分子运输机制。这阻碍了我们对DHA被输送到大脑和排除了用于神经治疗干预的基于结构的LPC前药设计。在这里，我们提出了MFSD2A的第一个结构，它是一个内向开放的apo构象-in复合体，带有一个Fab to 增加成像粒子的大小并向其引入特征-使用单粒子冷冻确定电子显微镜的分辨率为3.45？与杜克国家大学(新加坡)的David Silver博士合作，作为MSFD2A生理学领域的先驱，我们开发了生化分析来探索基于结构的 MSFD2A在细胞、溶液和蛋白脂质体中的功能假说。我们建议确定 MSFD2A及其配体的络合物和外向构象的结构，并使用我们的新的开发了生化分析来探索和充分了解这些结构的分子特征。这个这一应用的结果将阐明MSFD2A介导的分子机制 LPCDHA运输，并将提供有关DHA如何进入大脑的信息。此外，这项研究还有可能为合理设计神经疗法奠定基础，MFSD2A为通过血脑屏障传递。