TRANSFORMATIVE LIPID EXCHANGE APPROACHES TO STUDY MEMBRANE ORGANIZATION

研究膜组织的变革性脂质交换方法

• 批准号: 10591609
• 负责人: Erwin London
• 金额: $ 57.62万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10591609
• 关键词: 2019-nCoV; Antibodies; Biological Assay; Cell membrane; Cells; Cholesterol; Cyclodextrins; Disease; Environment; Erythrocytes; Fatty acid glycerol esters; GTP-Binding Proteins; Grant; Immune; In Situ; Insulin Receptor; Lipids; Liquid substance; Mammalian Cell; Membrane; Membrane Lipids; Membrane Proteins; Metabolic; Methods; Modeling; Mutation; Phospholipids; Physiological; Preparation; Signal Transduction; Sphingolipids; Sterols; Structure; Testing; Thick; Unsaturated Fats; Vaccines; Vesicle; Vesicular stomatitis Indiana virus; Viral; Virus; design; immunogenicity; improved; lipid structure; monolayer; novel; novel strategies; protein function; receptor; receptor function; response; segregation; uptake

We aim to understand biomembrane structure/function with the aid of a lab-achieved breakthrough in control of membrane phospholipid and sphingolipid composition via cyclodextrin-catalyzed lipid exchange. Lipid exchange is generally efficient (80-100%), and permits preparation of lipid vesicles mimicking natural membranes closely in terms of lipid asymmetry, the difference in the lipid composition in the inner and outer lipid monolayers (leaflets). The lab has now extended this method to control lipid composition of plasma membrane outer leaflets in mammalian cells (without harming them), and without disturbing membrane sterol. The method is being applied to solve long-standing issues of membrane organization and function. We proposed in 1994 what remains the working model: that membrane domains form in cells due to segregation of sphingolipid-cholesterol rich liquid ordered (Lo) domains from unsaturated lipid rich liquid disordered domains. In the last grant period, using lipid exchange to prepare asymmetric membranes, we found how lipid structure in each leaflet controls when there is overall induction or suppression of Lo domains. With physiologically-relevant lipids, we found that Lo domain formation is greatly enhanced by loss of lipid asymmetry, and confirmed this is also true in natural plasma membrane vesicles. This is important because transient loss of lipid asymmetry occurs during signal transduction. In the next grant period we propose to test the hypothesis that Lo domain formation is induced by loss of lipid asymmetry in intact cells by using red blood cells, which have a number of advantages including only a single membrane, known lipid asymmetry, known methods to control lipid asymmetry in situ, and facile use for lipid exchange. We will then extend studies to metabolically-active cells to see if loss of lipid asymmetry stabilizes Lo domains during antibody-triggered signal transduction in immune RBL-2H3 cells, and defining the importance of Lo domains by exchange using lipids that enhance or inhibit Lo domain formation. In the last grant period we also used lipid exchange to show the ability to form Lo domains is necessary and sufficient for a lipid to support insulin receptor function. We proposed the hypothesis that localization in Lo domain, which are thick, decreases insulin receptor transmembrane helix tilt, activating autophosphorylation. We will test this hypothesis by defining receptor domain localization before and after activation, and by mutations that lengthen or shorten its transmembrane segment, to see if predicted activity changes are observed. We also propose to test a novel competition assay for defining receptor domain localization. Finally, we propose to extend lipid exchange to membrane enveloped viruses. A range of lipids will be exchanged into the envelope of pseudovirus with SARS CoV-2 spike or VSV-G proteins to see how viral lipids/domains modulate cellular uptake. These studies may help design of improved vaccines.

我们的目标是了解生物膜的结构/功能的帮助下，实验室实现的突破， 通过环糊精催化的脂质控制膜磷脂和鞘脂组成 交易所脂质交换通常是有效的（80-100%），并允许制备脂质囊泡 在脂质不对称性方面密切模仿天然膜， 在内部和外部脂质单层（小叶）中的组合物。实验室现在已经将这种方法扩展到 控制哺乳动物细胞中质膜外小叶的脂质组成（不损害 他们），而不干扰膜甾醇。该方法正在应用于解决长期存在的 膜组织和功能的问题。我们在1994年提出了一个工作模式： 细胞中膜结构域的形成是由于富含鞘脂-胆固醇的液体的有序分离 (Lo)从不饱和脂质丰富的液体无序结构域的结构域。在上一个赠款期间，使用脂质 交换制备不对称膜，我们发现每个小叶中的脂质结构如何控制 当存在Lo结构域的总体诱导或抑制时。与生理相关的脂质，我们 发现Lo结构域的形成因脂质不对称性的丧失而大大增强，并证实这是 在天然质膜囊泡中也是如此。这一点很重要，因为脂质的短暂损失 在信号转导期间发生不对称。在下一个资助期内，我们将检验这一假设 Lo结构域的形成是通过使用红细胞在完整细胞中丧失脂质不对称性而诱导的， 其具有许多优点，包括仅单一膜、已知的脂质不对称性、已知的脂质不对称性和已知的脂质不对称性。 原位控制脂质不对称性的方法，以及用于脂质交换的简便方法。然后我们将扩展 对代谢活性细胞的研究，以观察脂质不对称性的丧失是否在代谢期间稳定Lo结构域。 在免疫RBL-2 H3细胞中抗体触发的信号转导，并定义Lo 通过使用增强或抑制Lo结构域形成的脂质进行交换来形成Lo结构域。在上一个资助期内 我们还使用脂质交换来显示形成Lo结构域的能力对于一个人来说是必要的和足够的。 脂质支持胰岛素受体功能。我们提出假设，在Lo域的定位， 其较厚，降低胰岛素受体跨膜螺旋倾斜，激活自身磷酸化。 我们将通过定义激活前后的受体结构域定位， 延长或缩短其跨膜片段的突变，以观察预测的活性变化是否 观察我们还建议测试一种新的竞争测定定义受体域定位。 最后，我们建议将脂质交换扩展到膜包膜病毒。一系列脂质将被 交换到具有SARS CoV-2刺突或VSV-G蛋白的假病毒的包膜中， 病毒脂质/结构域调节细胞摄取。这些研究可能有助于设计改进的疫苗。