VISUALIZING LOCALIZATION & TRANSLOCATION OF THREE TYPES OF PHOSPHOLIPASE A2

可视化本地化

• 批准号: 7358028
• 负责人: EDWARD A DENNIS
• 金额: $ 0.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7358028
• 关键词: VISUALIZING; LOCALIZATION; TRANSLOCATION; THREE; TYPES

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phospholipase A2 (PLA2) plays important roles in diverse cellular responses including fundamental metabolism and signal transduction by generating lysophospholipids and fatty acids such as arachidonic acid (AA), which is the precursor of eicosanoids. To date, many subtypes of mammalian PLA2s have been identified and classified into subgroups. In this study, we focus on three subtypes of them; the cytosolic group IV PLA2 (cPLA2), the secretory group V (sPLA2), and the cytosolic Ca2+independent group VI PLA2 (iPLA2). In the previous year (2003), we examined the secreted form of PLA2 and conducted a series of studies on its activation in macrophages following chronic exposure to lipopolysaccharide. Because sPLA2 is a secreted enzyme, it has been suggested that after cellular stimulation, it must be released to the extra-cellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. Using confocal laser scanning microscopy and GFP-tagged versions of sPLA2, we found that chronic exposure to lipopolysaccharide results in sPLA2 being associated with caveolin-2- containing granules close to the perinuclear region. This association is blocked by heparin (a cell-impermeable compound with high affinity for sPLA2), suggesting that the granules are formed by the internalization of sPLA2 previously associated with the outer cell surface. Perinuclear localization is not observed if the cells are treated with the group IV PLA2 inhibitor methyl arachidonyl fluorophosphonate, further indicating the important role played by cPLA2 in the activation process. These studies provided evidence that the encapsulation of sPLA2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to sPLA2 and COX-2 (cyclo-oxygenase-2) for efficient prostaglandin synthesis. Results from this project have been published on the Journal of Biological Chemistry in 2003 [Balboa et al., ¿Localization of group V phospholipase A2 in caveolin-enriched granules in activated P388D1 macrophage-like cells.¿(2003). Journal of Biological Chemistry, 278 (48), 48059-48065].

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。磷脂酶A2（PLA 2）通过产生溶血磷脂和脂肪酸如花生四烯酸（AA）（类花生酸的前体）在多种细胞反应中起重要作用，包括基本代谢和信号转导。迄今为止，已鉴定出哺乳动物PLA 2的许多亚型并将其分类为亚组。在这项研究中，我们专注于他们的三个亚型：细胞质组IV PLA 2（cPLA 2），分泌组V（sPLA 2），和细胞质钙离子非依赖组VI PLA 2（iPLA 2）。在前一年（2003年），我们检查了分泌形式的PLA 2，并进行了一系列的研究，其激活后，在巨噬细胞慢性暴露于脂多糖。由于sPLA 2是一种分泌型酶，因此有人提出，在细胞刺激后，它必须释放到细胞外介质中并与外膜重新结合以从磷脂中释放花生四烯酸。使用共聚焦激光扫描显微镜和GFP标记的sPLA 2的版本，我们发现，长期暴露于脂多糖的结果sPLA 2与小窝蛋白-2含有颗粒接近核周区域。这种结合被肝素（一种对sPLA 2具有高亲和力的细胞不可渗透的化合物）阻断，表明颗粒是通过先前与细胞外表面结合的sPLA 2的内化形成的。如果用第IV组PLA 2抑制剂甲基花生四烯酰氟膦酸酯处理细胞，则未观察到核周定位，进一步表明cPLA 2在活化过程中发挥的重要作用。这些研究提供的证据表明，在细胞活化期间，sPLA 2包封到颗粒中将酶带到核周包膜，在那里它可能更接近sPLA 2和考克斯-2（环氧合酶-2），用于有效的前列腺素合成。该项目的结果已于2003年发表在Journal of Biological Chemistry上[Balboa等人，<$在活化的P388 D1巨噬细胞样细胞中，V组磷脂酶A2在小窝蛋白富集颗粒中的定位。<$（2003年）的报告。Journal of Biological Chemistry，278（48），48059-48065]。