VISUALIZING LOCALIZATION & TRANSLOCATION OF THREE TYPES OF PHOSPHOLIPASE A2

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。磷脂酶A2(PLA2)通过产生溶血磷脂和花生四烯酸(AA)等脂肪酸，在包括基础代谢和信号转导在内的多种细胞反应中发挥重要作用，花生四烯酸是二十烷酸的前体。到目前为止，哺乳动物PLA2的许多亚型已经被识别并分类为亚组。在本研究中，我们重点研究了其中的三种亚型：胞内IV组PLA2(CPLA2)、分泌组V(SPLA2)和胞内钙非依赖性组VI组PLA2(IPLA2)。在前一年(2003)，我们检测了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，有人认为，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，从磷脂中释放花生四烯酸。使用激光共聚焦扫描显微镜和GFP标记的sPLA2版本，我们发现长期暴露于内毒素导致sPLA2与靠近核周区域的含有小窝蛋白-2的颗粒有关。这种联系被肝素(一种对sPLA2具有高亲和力的细胞不渗透化合物)阻断，这表明颗粒是由先前与细胞外表面相关的sPLA2内化形成的。如果用IV组PLA2抑制剂花生四烯基氟磷酸盐处理细胞，则没有观察到核周定位，进一步表明cPLA2在激活过程中发挥了重要作用。这些研究提供的证据表明，在细胞激活过程中，sPLA2被包裹成颗粒将酶带到核周膜，在那里它可能更接近于sPLA2和COX-2(环氧合酶-2)，从而有效地合成前列腺素。该项目的结果已发表在2003年的《生物化学杂志》上[Balboa等人，激活的P388D1巨噬细胞样细胞中小窝蛋白强化颗粒中V组磷脂酶A2的定位(2003)。生物化学杂志，278(48)，48059-48065]。

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].