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].

该主题项目是利用NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。磷脂酶A2（PLA2）通过产生溶血磷脂和脂肪酸（例如花生四烯酸（AA）），这是eicosanoids的前体。迄今为止，已经确定了许多哺乳动物PLA2的亚型，并将其分类为亚组。在这项研究中，我们专注于其中的三个亚型。胞质群IV PLA2（CPLA2），秘密组V（SPLA2）和胞质CA2+独立组VI PLA2（IPLA2）。在上一年（2003年）中，我们检查了PLA2的分泌形式，并在长期暴露于脂多糖后对巨噬细胞的激活进行了一系列研究。由于SPLA2是一种分泌的酶，因此有人建议在细胞刺激后，必须将其释放到细胞外培养基中，并与外膜重新缔合以从磷脂中释放蛛网膜含量。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导致SPLA2与caveolin-2-含有围核区域的颗粒相关。该关联被肝素（一种可渗透性的化合物对SPLA2具有高亲和力）阻塞，表明颗粒是由先前与外部细胞表面相关的SPLA2的内在化形成的。如果将细胞用IV组PLA2抑制剂甲基芳基膦酸酯处理，未观察到核周定位，进一步表明CPLA2在激活过程中所起的重要作用。这些研究提供了证据表明，在细胞激活期间，将SPLA2封装在颗粒中，将酶带到了核周包膜中，在细胞激活中，它可能更接近SplA2和Cox-2（环氧酶-2），以进行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，在可活化的p388d1巨噬细胞样细胞中富含小窝蛋白添加的颗粒中的V组V磷脂酶A2的定位。生物化学杂志，278（48），48059-48065]。