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: 9883010
• 负责人: Erwin London
• 金额: $ 54.12万
• 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 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9883010
• 关键词: Bacteria; Bacterial Infections; Borrelia burgdorferi; Cell membrane; Cells; Characteristics; Chemical Structure; Cholesterol; Coupling; Cyclodextrins; Detection; Disease; Drug resistance; Electron Microscopy; Fluorescence; Fluorescence Resonance Energy Transfer; Helicobacter pylori; In Vitro; Knowledge; Lipids; Liquid substance; Lyme Disease; Malignant Neoplasms; Mammalian Cell; Membrane; Membrane Lipids; Methods; Modeling; Molecular; Organism; Phospholipids; Plasma Exchange; Preparation; Property; Proteins; Public Health; Signal Transduction; Sphingolipids; Staphylococcus aureus; Sterols; Structure; Testing; Time; Ulcer; Unsaturated Fats; Vesicle; Visualization; combat; human disease; in vivo; insight; methicillin resistant Staphylococcus aureus; method development; novel strategies; pathogenic bacteria; physical property; resistant strain; segregation

This project aims to understand biomembrane structure and function with the aid of a recent lab-achieved breakthrough in control of membrane phospholipid and sphingolipid composition via cyclodextrin-catalyzed lipid exchange. This permits preparation of lipid vesicles mimicking natural membranes closely in terms of both lipid composition and, for the first time, lipid asymmetry, the difference in the lipid composition in the inner and outer lipid layers characteristic of many natural membranes. The method is also being extended to control of lipid composition in living cells. These methods are being applied to solve long-standing issues of membrane domain formation. We and collaborator Dr. Deborah Brown proposed in 1994 what remains the working model in the field: cell membrane domains form due to segregation of sphingolipid-cholesterol rich liquid ordered (Lo) domains from unsaturated lipid rich liquid disordered domains. Although such domains are readily observed in artificial lipid vesicles, and careful studies document their presence in living cells, domains remain controversial and poorly characterized. This project will use lipid exchange to overcome roadblocks to progress in studies of membrane domains. First, studies using asymmetric lipid vesicles that mimic cell membranes much more closely than the symmetric vesicles employed in the past will define the rules governing domain properties and formation by lipids and proteins. This includes testing the long-standing hypothesis that lipid-induced signal transduction across membranes can result from coupling between physical properties of lipids in the inner and outer lipid layers of a membrane. Knowledge gained from these studies will reveal how to manipulate lipids and proteins in cells to control domain formation and protein association with domains, and thus how to explore their function. Second, domains will be studied with more tractable living systems and methods. We found that the cholesterol-containing bacterium Borrelia burgdorferi, the causative agent of Lyme disease, has domain size sufficient for visualization by electron microscopy and facile fluorescence domain detection using FRET, plus accessibility to altering sterol chemical structure to allow controlled modulation of domain formation. This made it possible to unequivocally identify bacterial Lo domains in vitro and in vivo. Studies will be extended to other pathogenic bacteria likely to contain Lo domains: Helicobacter pylori, the cause of ulcers and some cancers, and S. aureus, drug-resistant strains of which (MRSA) are a major public health threat. Studies will define the properties of bacterial domains, the principles behind their formation, and potential biomedical implications. Studies will then be extended to mammalian cells taking advantage of our discovery of a cyclodextrin that can fully exchange plasma membranes outer leaflet lipids with exogenous phospholipid and sphingolipid without disturbing cell sterols. Using this, how altering lipids modulate domain formation, properties, and function will be defined. This method will also be used to investigate plasma membrane lipid asymmetry and its function. Further development of the method will broadly impact biomembrane studies.

该项目的目的是了解生物膜的结构和功能的帮助下，最近的实验室实现 通过环糊精催化控制膜磷脂和鞘脂组成的突破 脂质交换这允许制备脂质囊泡，其在两个方面都紧密地模仿天然膜。 脂质组成和，第一次，脂质不对称，在内部和内部的脂质组成的差异， 许多天然膜的外层脂质层。该方法还被扩展到控制 活细胞中的脂质成分。这些方法正在被应用于解决长期存在的问题，膜 域形成。我们和合作者黛博拉·布朗博士在1994年提出了一个仍然有效的模型 在该领域：由于鞘脂-胆固醇富集液体的分离而形成细胞膜结构域有序（Lo） 从不饱和脂质丰富的液体无序结构域的结构域。尽管这些结构域在 人工脂质囊泡，仔细的研究证明它们存在于活细胞中，领域仍然存在争议 并且特征不明显。该项目将使用脂质交换来克服研究进展的障碍， 膜结构域首先，使用不对称脂质囊泡的研究， 比过去使用的对称囊泡更接近地将定义支配域性质的规则， 由脂质和蛋白质形成。这包括测试长期存在的假设，即脂质诱导的信号 跨膜转导可由内部脂质的物理性质与内部脂质的物理性质之间的耦合引起， 膜的外部脂质层。从这些研究中获得的知识将揭示如何操纵脂质， 细胞中的蛋白质来控制结构域的形成和蛋白质与结构域的结合，从而探索 它们的功能。第二，将用更易处理的生活系统和方法来研究领域。我们发现 莱姆病的病原体，含胆固醇的伯氏疏螺旋体， 其尺寸足以通过电子显微镜观察和使用FRET的容易的荧光结构域检测， 加上改变甾醇化学结构的可及性，以允许域形成的受控调节。这 使得在体外和体内明确鉴定细菌Lo结构域成为可能。研究将扩展到 其他致病菌可能含有Lo域：幽门螺杆菌，溃疡的原因和一些 癌和S.金黄色葡萄球菌，耐药菌株（MRSA）是一个主要的公共卫生威胁。研究将 定义细菌域的特性，它们形成背后的原理，以及潜在的生物医学 含义。研究将扩展到哺乳动物细胞利用我们的发现， 环糊精可完全交换质膜外小叶脂质与外源性磷脂， 鞘脂而不干扰细胞固醇。利用这一点，改变脂质如何调节结构域的形成， 属性和功能将被定义。该方法也可用于研究细胞膜脂质 不对称性及其功能。该方法的进一步发展将广泛影响生物膜研究。