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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。磷脂酶 A2 (PLA2) 通过生成溶血磷脂和脂肪酸（如花生四烯酸 (AA)，类二十烷酸的前体），在多种细胞反应中发挥重要作用，包括基础代谢和信号转导。迄今为止，哺乳动物 PLA2 的许多亚型已被鉴定并分为亚组。在这项研究中，我们重点关注其中的三个亚型；细胞质 IV 族 PLA2 (cPLA2)、分泌族 V (sPLA2) 和细胞质 Ca2+ 独立 VI 族 PLA2 (iPLA2)。去年（2003年），我们检查了PLA2的分泌形式，并对其在长期暴露于脂多糖后在巨噬细胞中的激活进行了一系列研究。由于sPLA2是一种分泌酶，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，从磷脂中释放出花生四烯酸。使用共聚焦激光扫描显微镜和 sPLA2 的 GFP 标记版本，我们发现长期暴露于脂多糖会导致 sPLA2 与靠近核周区域的含有 Caveolin-2 的颗粒相关。这种关联被肝素（一种对 sPLA2 具有高亲和力的细胞不可渗透化合物）阻断，表明颗粒是通过先前与外细胞表面关联的 sPLA2 的内化形成的。如果用IV组PLA2抑制剂花生四烯基氟膦酸甲酯处理细胞，则不会观察到核周定位，进一步表明cPLA2在激活过程中发挥的重要作用。这些研究提供的证据表明，在细胞激活过程中，将 sPLA2 封装到颗粒中会将酶带到核周包膜，在那里它可能更接近 sPLA2 和 COX-2（环氧化酶-2），以实现有效的前列腺素合成。该项目的结果已于 2003 年发表在《生物化学杂志》上 [Balboa 等人，“活化 P388D1 巨噬细胞样细胞中富含小窝蛋白的颗粒中 V 族磷脂酶 A2 的定位”(2003)。生物化学杂志，278（48），48059-48065]。