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

该项目的重点是氧化磷脂的混合组装体的生物物理特性。 目的1与心、脑磷脂酰胆碱的关系及其作用机制的探讨 诱导人第V组(Hgv)和第IIa组(HgIIa)分泌型磷脂酶A2(SPLA2)活性。 已发表的工作和初步的Laurdan发射光谱显示OxPL和双层PL的混合物显示类脂 以OxPL富结构域或胶束和双层富PL结构域或小泡的形式组成的有序。 分离的物理化学基础被假设为(I)氢键介导的键 一个OxPL极性末端基团和另一个OxPL酯基，它们将OxPL聚集在一起形成OxPL 丰富的结构域；(Ii)倒锥形OxPL和锥形OxPL的分子形状差异 导致OxPL结构域的正曲率和双层PL的负曲率的双层类脂 分别为域。曲率应力最终导致富OxPL结构域以胶束的形式分离。 温度和脂质不饱和度增加了双层锥角，这是因为增加了链的流动性和 进一步突出形态差异，促进分流。氧磷酸盐1-棕榈酰基-2-戊二酰锡的混合物- 3-甘油-3-磷酸胆碱(羧基)和1-棕榈酰基-2-(5‘-氧代-戊酰基)-sn-甘油-3- 磷胆碱(乙醛端基)和心脏和脑PC脂类将用动态光法进行研究 集料尺寸的散射(DLS)和Laurdan荧光检测偏析。混合状态 预测为：均匀混合，含有OxPL和双层PL富含结构域的双层，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，但与 分离的结构域被简单地报告为小泡。利用Laurdan荧光激发的灵敏度 而利用对数正态分布的结构域脂间键发射和反卷积将是新的 更好地检测域的方法。该假说预测，分离将更加明显，当 端基是极性更强的羧基，而不是醛。众所周知，膜氧化是一种 引发前炎性sPLA2活性的主要原因。分离会刺激酶的活性，因为 高曲率OxPL结构域或胶束是很容易获得的底物。截断中的端组 OxPL的尾部是亲水性的，指向使脂质突出的界面，进一步增加了它的 可访问性。由于这些原因，HGV sPLA2能使PC膜发生水解性反应，从而提高其活性。香港政府资讯科技署 SPLA2不与PC膜结合，也不能对PC膜进行水解。然而，带电的界面存在 OxPL的截短末端基团创建了一个带电界面，该酶可以结合到该界面上并进行刺激 水解液。目前使用纯PC双层膜的研究将解决OxPL是否刺激 SPLA2与磷脂酰丝氨酸或其他带电的双层脂类无关。酶的活性将是 用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。

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.