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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.

摘要含有主要易化因子超家族结构域2A（MFSD 2A）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内高度表达，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包含多达 20%的脑脂质。尽管如此，大脑不能重新合成DHA和MFSD 2A介导的转运是大脑获取DHA的主要途径。小鼠MFSD 2A KO模型表现出严重的脑DHA 缺乏症和广泛性小头畸形，以及具有纯合功能丧失突变的人类患者， MFSD 2A表现为严重的小头畸形和发育迟缓。先前基于细胞的研究已经证明MFSD 2A以如下形式转运DHA：溶血磷脂酰胆碱（LPCDHA），而不是未酯化的脂肪酸，在Na+依赖性的方式。类似于所有其他MFS家族成员，MFSD 2A具有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，MFSD 2A运输溶血脂，提高了独特运输的可能性机制据推测，MFSD 2A通过以下途径转运单酰基链脂质穿过膜：在膜小叶之间翻转LPC，基本上充当“翻转酶”。了解MFSD 2A- 介导的转运蛋白具有帮助药物递送到大脑的潜力。概念验证研究表明，与LPC共价连接的小分子可以通过MFSD 2A转运，这可能提供了一种新的这是一个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，还没有已知的MFSD 2A的原子分辨率结构，并且对其结构的理解有限。分子输运机制这阻碍了我们对DHA被输送到大脑中的人的理解，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，我们呈现了MFSD 2A的第一个结构，其呈载脂蛋白向内开放构象-与Fab复合，增加成像颗粒的尺寸并向其引入特征-使用单颗粒低温测定电子显微镜至3.45 μ m分辨率。与DUKE-NUS（新加坡）的大卫银博士合作，作为MSFD 2A生理学领域的先驱，我们已经开发了生物化学测定来探测基于结构的 MSFD 2A在细胞、溶液和蛋白脂质体中的功能假说。我们建议确定 MSFD 2A的结构与其配体复合并处于向外的构象，并使用我们的新开发了生物化学分析来探测和充分了解这些结构的分子特征。的本申请产生的结果将阐明MSFD 2A介导的 LPCDHA运输并提供有关DHA如何进入大脑的信息。此外，这项研究有可能为合理设计“劫持”MFSD 2A的神经治疗奠定基础。通过血脑屏障递送。