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:
- 金额:$ 24.3万
- 依托单位:
- 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:
- 关键词:AffinityBindingBiochemicalBiological AssayBiophysicsBloodBlood - brain barrier anatomyBlood-Retinal BarrierBrainCellsCellular AssayCollaborationsComplementComplexCryoelectron MicroscopyDataData CollectionDetergentsDevelopmentDevelopmental Delay DisordersDocosahexaenoic AcidsDrug Delivery SystemsEndothelial CellsExhibitsEyeFab ImmunoglobulinsFamily memberFatty AcidsFoundationsHeadHumanImageIntegral Membrane ProteinInterventionKnock-outLigandsLinkLipidsLysophosphatidylcholinesMeasuresMediatingMembraneMethodsMicrocephalyModelingMolecularMolecular ConformationMusN DomainNatureOmega-3 Fatty AcidsPatientsPhysiologicalPhysiologyPolyunsaturated Fatty AcidsPositioning AttributePreparationProdrugsRecombinantsResearchResolutionRouteSalmonSamplingSilverSingaporeStructureSystemTestingTransmembrane DomainTransport ProcessWateranalogbasebrain celldesignexperimental studyfatty acid transportloss of function mutationmembernanodisknovelparticleprotein transportproteoliposomesrational designreconstitutionsmall moleculestructural biologyuptake
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。
项目成果
期刊论文数量(1)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Structural Insights into Transporter-Mediated Drug Resistance in Infectious Diseases.
- DOI:10.1016/j.jmb.2021.167005
- 发表时间:2021-08-06
- 期刊:
- 影响因子:5.6
- 作者:Kim J;Cater RJ;Choy BC;Mancia F
- 通讯作者:Mancia F
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Filippo Mancia其他文献
Filippo Mancia的其他文献
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{{ truncateString('Filippo Mancia', 18)}}的其他基金
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
- 批准号:
10156975 - 财政年份:2020
- 资助金额:
$ 24.3万 - 项目类别:
Structural basis of integral membrane enzyme function
完整膜酶功能的结构基础
- 批准号:
10609459 - 财政年份:2019
- 资助金额:
$ 24.3万 - 项目类别:
Structural basis of integral membrane enzyme function
完整膜酶功能的结构基础
- 批准号:
9921455 - 财政年份:2019
- 资助金额:
$ 24.3万 - 项目类别:
Structural basis of integral membrane enzyme function
完整膜酶功能的结构基础
- 批准号:
10582102 - 财政年份:2019
- 资助金额:
$ 24.3万 - 项目类别:
Structural basis of integral membrane enzyme function
完整膜酶功能的结构基础
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10393522 - 财政年份:2019
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