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.

本项目的重点是氧化磷脂混合组装体的生物物理表征 目的：探讨OxPL与心、脑磷脂酰胆碱（PC）脂质的关系，并阐明OxPL的作用机制 在Aim 2中诱导人V型（hgV）和IIa型（hgIIa）分泌型磷脂酶A2（sPLA 2）活性。 已发表的工作和初步劳登发射光谱的混合物OxPL和双层PL显示脂质 富OxPL结构域或胶束和双层富PL结构域或囊泡形式的组成有序化。 分层的物理化学基础被假设为（i）氢键介导的键合， 一个OxPL极性端基和另一个OxPL酯基，其将OxPL结合在一起形成OxPL 丰富的领域;（ii）反锥形OxPL和锥形OxPL之间的分子形状差异 双层脂质，其诱导OxPL结构域中的正曲率和双层PL中的负曲率 域分别。曲率应力最终导致富含OxPL的结构域作为胶束分离。 温度和脂质不饱和度增加了双层锥角，因为增加了链的流动性， 进一步突出形状差异并促进分层。OxPL 1-棕榈酰基-2-戊二酰基-sn- 甘油基-3-磷酸胆碱（羧基末端基团）和1-棕榈酰基-2-（5 '-氧代-戊酰基）-sn-甘油基-3- 磷酸胆碱（醛端基）和心脏和大脑PC脂质将通过动态光研究 散射（DLS）的聚集体大小和劳登荧光检测偏析。混合状态 预测的是：均匀混合，双层与OxPL和双层PL丰富的结构域，共存的双层脂质 囊泡和OxPL胶束。胶束/囊泡共存很容易通过DLS检测，但混合双层与 分离的结构域被简单地报道为囊泡。利用Laurdan荧光激发的灵敏度 和发射到域中的脂质间键合和使用对数正态分布的去卷积将是新颖的 更好地检测域。该假说预测，当 端基是极性更大的羧基而不是醛。膜氧化法是一种 引发促炎性sPLA 2活性的主要原因。分离刺激酶活性，因为 高曲率OxPL域或胶束是高度可接近的底物。在截断的 OxPL的尾部是亲水性的，并指向界面，使脂质突出，进一步增加其 可访问性。由于这些原因，HgV sPLA 2水解PC膜并增加其活性。HgIIA sPLA 2不结合PC膜，也不水解PC膜。然而，界面存在带电的 OxPL的截短的末端基团产生带电界面，该酶可以与之结合，并刺激 水解目前的研究使用纯PC双层将解决的问题，是否OxPL刺激 与磷脂酰丝氨酸或其他带电双层脂质暴露无关。酶活性将是 通过pH-Stat方法测量，以确定增加的活性和结构域形成之间的相关性。