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）：分泌型磷脂酶 A2 (sPLA2) 与脂质双层结合并催化磷脂水解。正常情况下，细胞会抵抗酶的作用，但在细胞凋亡或创伤过程中它们会变得敏感。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，当细胞对 sPLA2 敏感时，有序和无序脂质域之间的界限就会增殖。假设在这些边界处减少相邻磷脂之间的有利相互作用，可以通过促进磷脂迁移到酶活性位点来增强 sPLA2 活性。该提案将这些研究扩展到有核细胞，并测试激素刺激细胞凋亡期间的假设。将提出四个问题。 1) 细胞凋亡过程中膜顺序是否发生变化？它们是否会减少磷脂与邻近磷脂的相互作用？ 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增强吸附、脂质迁移至吸附酶的活性位点，或两者兼而有之？ 3）这个假设理论上可行吗？ 4) 这些机制如何应用于各种类型的哺乳动物 sPLA2？为了回答这些问题，将使用六种一般程序来研究地塞米松刺激细胞凋亡期间淋巴瘤细胞的变化。首先，将使用膜探针 laurdan 通过荧光光谱和显微镜检查膜物理特性的变化。其次，通过部花青 540 的荧光和测量白蛋白从细胞膜提取荧光磷脂的速率来评估磷脂-邻近相互作用的强度。第三，将在各种酶浓度下测定膜水解动力学，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 重复进行。第四，通过计算机模拟对假设进行理论上的评估。第五，将测量 sPLA2 与细胞膜表面的结合。第六，通过测量sPLA2提取磷脂的速率来评估磷脂迁移至结合酶的活性位点的能力。

项目成果

Relationship between membrane physical properties and secretory phospholipase A2 hydrolysis kinetics in S49 cells during ionophore-induced apoptosis.

离子载体诱导细胞凋亡过程中 S49 细胞膜物理特性与分泌型磷脂酶 A2 水解动力学之间的关系。

• 发表时间: 2007-10-01
• 影响因子: 3.4
• 作者: Bailey, Rachel W;Olson, Erin D;Vu, Mai P;Brueseke, Taylor J;Robertson, Leslie;Christensen, Ryan E;Parker, Kristen H;Judd, Allan M;Bell, John D
• 通讯作者: Bell, John D

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。

• 发表时间: 2006-05
• 作者: Vest, Rebekah;Wallis, Rachel;Jensen, Lauren B;Haws, Andrea C;Callister, Joseph;Brimhall, Brent;Judd, Allan M;Bell, John D
• 通讯作者: Bell, John D

A new hat for an old enzyme: waste management.

旧酶的新帽子：废物管理。

• 发表时间: 2006-11
• 作者: Brueseke, Taylor J;Bell, John D
• 通讯作者: Bell, John D

Kinetic evaluation of cell membrane hydrolysis during apoptosis by human isoforms of secretory phospholipase A2.

人分泌型磷脂酶 A2 亚型在细胞凋亡过程中细胞膜水解的动力学评估。

• 发表时间: 2010-04-02
• 作者: Olson, Erin D;Nelson, Jennifer;Griffith, Katalyn;Nguyen, Thaothanh;Streeter, Michael;Wilson;Gelb, Michael H;Judd, Allan M;Bell, John D
• 通讯作者: Bell, John D

Effects of cholesterol on physical properties of human erythrocyte membranes: impact on susceptibility to hydrolysis by secretory phospholipase A2.

胆固醇对人红细胞膜物理特性的影响：对分泌性磷脂酶 A2 水解敏感性的影响。

• 发表时间: 2008-04-15
• 影响因子: 3.4
• 作者: Heiner, Anne L;Gibbons, Elizabeth;Fairbourn, Jeremy L;Gonzalez, Laurie J;McLemore, Chisako O;Brueseke, Taylor J;Judd, Allan M;Bell, John D
• 通讯作者: Bell, John D