Action of Phospholipase A2 on Apoptotic Cells

磷脂酶A2对凋亡细胞的作用

• 批准号: 6898132
• 负责人: JOHN D BELL
• 金额: $ 22.5万
• 依托单位: BRIGHAM YOUNG UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6898132
• 关键词: active sites; adsorption; albumins; apoptosis; biophysics; cell line; chemical kinetics; computer simulation; dexamethasone; enzyme mechanism; enzyme model; erythrocytes; fluorescence microscopy; fluorescent dye /probe; human tissue; hydrolysis; lipid bilayer membrane; lymphocyte; phospholipase A2; phospholipids; solvent extraction; statistics /biometry

DESCRIPTION (provided by applicant): Secretory phospholipase A2 (sPLA2) binds to lipid bilayers and catalyzes hydrolysis of phospholipids. Normally, cells resist the enzyme's action, but they become susceptible during apoptosis or trauma. Hydrolysis at the membrane surface apparently requires two steps: enzyme adsorption to the membrane surface followed by migration of phospholipids from their normal bilayer position up into the enzyme's active site. Experiments using erythrocytes as a model suggest that when cells become susceptible to sPLA2, boundaries between domains of ordered and disordered lipids proliferate. Reduction of favorable interactions among neighboring phospholipids at those boundaries is hypothesized to enhance sPLA2 activity by facilitating migration of phospholipids into the enzyme active site. This proposal will extend these studies to nucleated cells and test the hypothesis during hormone-stimulated apoptosis. Four questions will be asked. 1) Do changes in membrane order occur during apoptosis and do they reduce phospholipids-neighbor interactions? 2) How does apoptosis increase susceptibility to sPLA2; does it promote enhanced adsorption of the enzyme to the membrane surface, migration of lipids into the active site of the adsorbed enzyme, or both? 3) Is the hypothesis theoretically feasible? 4) How do these mechanisms apply to the various types of mammalian sPLA2? To answer these questions, six general procedures will be used to study changes in lymphoma cells during apoptosis stimulated by dexamethasone. First, alterations to membrane physical properties will be examined by fluorescence spectroscopy and microscopy using the membrane probe laurdan. Second, the strength of phospholipid-neighbor interactions will be assessed by the fluorescence of merocyanine 540 and by measuring the rate at which albumin extracts fluorescent phospholipids from the cell membrane. Third, the kinetics of membrane hydrolysis will be assayed at various enzyme concentrations and mathematically analyzed in the context of the two-step model described above. These experiments will be repeated using various forms of mammalian sPLA2. Fourth, the hypothesis will be evaluated theoretically by computer simulations. Fifth, the binding of sPLA2 to the surface of the cell membranes will be measured. Sixth, the ability of phospholipids to migrate to the active site of bound enzyme will be assessed by measuring the rate of extraction of phospholipids by sPLA2.

描述（由申请人提供）：分泌磷脂酶A2（SPLA2）与脂质双层结合，并催化磷脂的水解。通常，细胞抵抗酶的作用，但在凋亡或创伤期间它们变得易感性。膜表面的水解显然需要两个步骤：酶吸附到膜表面，然后磷脂从其正常双层位置迁移到酶的活性位点。使用红细胞作为模型的实验表明，当细胞易受SPLA2的敏感时，有序脂质和无序脂质域之间的边界增殖。假设在这些边界处相邻磷脂之间的有利相互作用的降低可以通过促进磷脂迁移到酶活性位点来增强SPLA2活性。该提议将将这些研究扩展到核细胞，并在激素刺激的凋亡过程中检验假设。将问四个问题。 1）在凋亡过程中，膜顺序的变化是否会减少磷脂 - 邻居相互作用？ 2）细胞凋亡如何增加对SPLA2的敏感性；它是否促进酶对膜表面的吸附，将脂质迁移到吸附酶的活性位点，或两者兼而有之？ 3）假设理论上是可行的吗？ 4）这些机制如何适用于各种类型的哺乳动物SPLA2？为了回答这些问题，将使用六个一般程序来研究地塞米松刺激的凋亡过程中淋巴瘤细胞的变化。首先，使用膜探针Laurdan，将通过荧光光谱和显微镜检查膜物理特性的改变。其次，将通过梅罗糖苷540的荧光来评估磷脂 - 氯相互作用的强度，并通过测量白蛋白从细胞膜中提取荧光磷脂的速率来评估。第三，将以各种酶浓度分析膜水解的动力学，并在上述两步模型的背景下进行数学分析。这些实验将使用各种形式的哺乳动物SPLA2重复。第四，该假设将通过计算机模拟从理论上进行评估。第五，将测量SPLA2与细胞膜表面的结合。第六，将通过测量SPLA2的磷脂提取速率来评估磷脂迁移到结合酶的活性位点的能力。

项目成果

Use of steady-state laurdan fluorescence to detect changes in liquid ordered phases in human erythrocyte membranes.

使用稳态劳丹荧光检测人红细胞膜中液相有序相的变化。

• DOI: 10.1007/s00232-005-7008-6
• 发表时间: 2006
• 期刊: The Journal of membrane biology
• 作者: Vest,Rebekah;Wallis,Rachel;Jensen,LaurenB;Haws,AndreaC;Callister,Joseph;Brimhall,Brent;Judd,AllanM;Bell,JohnD
• 通讯作者: Bell,JohnD

Role of membrane oxidation in controlling the activity of human group IIa secretory phospholipase A(2) toward apoptotic lymphoma cells.

膜氧化在控制人 IIa 型分泌磷脂酶 A(2) 对凋亡淋巴瘤细胞活性中的作用。

• DOI: 10.1016/j.bbamem.2012.09.013
• 发表时间: 2013
• 期刊: Biochimica et biophysica acta
• 作者: Gibbons,Elizabeth;Nelson,Jennifer;Anderson,Lynn;Brewer,Kelly;Melchor,Stephanie;Judd,AllanM;Bell,JohnD
• 通讯作者: Bell,JohnD

Molecular details of membrane fluidity changes during apoptosis and relationship to phospholipase A(2) activity.

• DOI: 10.1016/j.bbamem.2012.08.024
• 发表时间: 2013-02
• 期刊: BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
• 影响因子: 3.4
• 作者: Gibbons, Elizabeth;Pickett, Katalyn R.;Streeter, Michael C.;Warcup, Ashley O.;Nelson, Jennifer;Judd, Allan M.;Bell, John D.
• 通讯作者: Bell, John D.

The influence of membrane physical properties on microvesicle release in human erythrocytes.

• DOI: 10.1186/1757-5036-2-7
• 发表时间: 2009-08-24
• 期刊: PMC biophysics
• 作者: Gonzalez LJ;Gibbons E;Bailey RW;Fairbourn J;Nguyen T;Smith SK;Best KB;Nelson J;Judd AM;Bell JD
• 通讯作者: Bell JD