Membrane Deformation and Mechanism of Stimulation of Phospholipase A2 by Oxidized Lipids

氧化脂质刺激磷脂酶A2的膜变形及机制

• 批准号: 9916778
• 负责人: Radha Ranganathan
• 金额: $ 10.88万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-02 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9916778
• 关键词: Aldehydes; Apoptosis; Automobile Driving; Binding; Biological Models; Brain; Cells; Charge; Cone; Endocytosis; Enzymes; Esters; Fatty Acids; Fluorescence; Goals; Heart; Human; Hydrogen Bonding; Hydrolysis; Lead; Lecithin; Lifting; Lipid Bilayers; Lipids; Liquid substance; Lysophospholipids; Measurement; Measures; Mediating; Membrane; Methods; Micelles; Modeling; Molecular; Oxides; Phosphatidylserines; Phospholipase A2; Phospholipid Interaction; Phospholipids; Phosphorylcholine; Play; Public Health; Publishing; Reaction; Reporting; Research; Role; Shapes; Signal Transduction; Stress; Surface; System; Tail; Temperature; Thin Layer Chromatography; Toxic effect; Vesicle; Water; Work; biophysical properties; chemical group; emission spectroscopy; enzyme activity; experimental study; fluidity; hydrophilicity; interfacial; laurdan; light scattering; molecular modeling; molecular shape; novel strategies; oxidation; oxidized lipid; segregation

The focus of this project is the biophysical characterization of mixed assemblies of oxidized phospholipids (OxPL) and heart and brain phosphatidylcholine (PC) lipids in Aim 1 and elucidation of the mechanism of OxPL induced human group V (hgV) and group IIa (hgIIa) secretory phospholipase A2 (sPLA2) activity in Aim 2. Published work and preliminary Laurdan emission spectroscopy on mixtures of OxPL and bilayer PL show lipid compositional ordering in the form of OxPL rich domains or micelles and bilayer PL rich domains or vesicles. A physicochemical basis for demixing is hypothesized to be (i) the hydrogen bond mediated bonding between one OxPL polar terminal group and another OxPL ester group which brings the OxPL together to form OxPL rich domains; (ii) the molecular shape difference between the inverse cone shaped OxPL and the conical bilayer lipid which induces positive curvature in the OxPL domains and negative curvature in the bilayer PL domains respectively. Curvature stresses eventually lead to separation of the OxPL rich domains as micelles. Temperature and lipid unsaturation increase the bilayer cone angle because of increased chain mobility and further accentuate the shape difference and promote demixing. Mixtures of the OxPL 1-palmitoyl-2-glutaryl-sn- glycero-3-phosphocholine (carboxyl terminal group) and 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3- phosphocholine (aldehyde terminal group) and heart and brain PC lipids will be investigated by Dynamic Light Scattering (DLS) for aggregate sizes and by Laurdan fluorescence to detect segregation. States of mixing predicted are: homogenous mixing, bilayer with OxPL and bilayer PL rich domains, coexisting bilayer lipid vesicles and OxPL micelles. Micelle / vesicle coexistence is readily detected by DLS, but mixed bilayers with segregated domains are reported simply as vesicles. Using the sensitivity of Laurdan fluorescence excitation and emission to inter-lipid bonding in domains and deconvolution using lognormal distributions will be novel approaches to better detect domains. The hypothesis predicts that demixing will be more pronounced when the terminal group is the more polar carboxyl rather than aldehyde. Membrane oxidation is known to be a leading cause in triggering proinflammatory sPLA2 activity. Segregation stimulates enzymatic activity because the high curvature OxPL domains or micelles are highly accessible substrates. The end group in the truncated tail of OxPL is hydrophilic and points to the interface making the lipid protrude, further increasing its accessibility. HgV sPLA2 hydrolyzes PC membranes and increases in its activity for these reasons. The hgIIA sPLA2 does not bind to and does not hydrolyze PC membranes. However interfacial presence of charged truncated tail end groups of OxPL creates a charged interface, to which this enzyme can bind, and stimulate hydrolysis. The present research using pure PC bilayers will resolve the question of whether OxPL stimulates sPLA2 irrespective of phosphatidylserine or other charged bilayer lipid exposure. Enzymatic activity will be measured by pH-Stat methods to determine correlation between increased activity and formation of domains.

该项目的重点是氧化磷脂混合组装体的生物物理表征 Aim 1 中的 (OxPL) 和心脑磷脂酰胆碱 (PC) 脂质以及 OxPL 机制的阐明 在目标 2 中诱导人 V 组 (hgV) 和 IIa 组 (hgIIa) 分泌型磷脂酶 A2 (sPLA2) 活性。 已发表的工作和 OxPL 和双层 PL 混合物的初步 Laurdan 发射光谱显示脂质 富含 OxPL 的结构域或胶束和双层 PL 丰富的结构域或囊泡形式的组成排序。 分层的物理化学基础被假设为 (i) 之间的氢键介导的键合 一个 OxPL 极性端基和另一个 OxPL 酯基，将 OxPL 结合在一起形成 OxPL 丰富的领域； (ii) 倒锥形OxPL和圆锥形之间的分子形状差异 双层脂质，在 OxPL 域中诱导正曲率，在双层 PL 中诱导负曲率 分别是域。曲率应力最终导致富含 OxPL 的结构域以胶束形式分离。 温度和脂质不饱和度会增加双层锥角，因为链的流动性和 进一步强调形状差异并促进分层。 OxPL 1-棕榈酰-2-戊二酰-sn-的混合物 甘油-3-磷酸胆碱（羧基末端基团）和 1-棕榈酰-2-(5'-氧代-戊酰)-sn-甘油-3- 磷酸胆碱（醛末端基团）以及心脏和大脑 PC 脂质将通过动态光进行研究 散射 (DLS) 测定聚集体大小，并通过 Laurdan 荧光检测分离。混合状态 预测为：均匀混合、具有 OxPL 的双层和富含双层 PL 的结构域、共存的双层脂质 囊泡和 OxPL 胶束。 DLS 很容易检测到胶束/囊泡共存，但混合双层 分离的结构域被简单地报告为囊泡。利用 Laurdan 荧光激发的灵敏度 域中脂质间键合的发射以及使用对数正态分布的反卷积将是新颖的 更好地检测域的方法。该假设预测，当 末端基团是极性更强的羧基而不是醛基。已知膜氧化是 触发促炎性 sPLA2 活性的主要原因。分离会刺激酶活性，因为 高曲率 OxPL 域或胶束是易于接近的基质。截断的末端基团 OxPL的尾部具有亲水性，指向界面，使脂质突出，进一步增加其 可达性。由于这些原因，HgV sPLA2 会水解 PC 膜并增加其活性。人类基因IIA sPLA2 不会结合 PC 膜，也不会水解 PC 膜。然而，界面存在带电 OxPL 的截短尾端基团创建了一个带电界面，该酶可以与其结合并刺激 水解。目前使用纯 PC 双层的研究将解决 OxPL 是否刺激的问题 sPLA2 与磷脂酰丝氨酸或其他带电双层脂质暴露无关。酶的活性将是 通过 pH-Stat 方法测量以确定活性增加和结构域形成之间的相关性。