Regulation of Phospholipase A2 Membrane Targeting

描述(由申请人提供)： IVA组胞浆磷脂酶A2α(cPLA2α)介导激动剂诱导的花生四烯酸的释放，花生四烯酸是二十烷类化合物的前体。这些强有力的脂质介质，包括前列腺素和白三烯，是许多正常生理过程所必需的，也促进急性炎症。由cPLA2α激活产生的二十烷基类化合物与急性肺损伤和肺纤维化的发生有关。本研究的目的是阐明cPLA2α靶向细胞膜的机制及其水解性的调节。cPLA2α受磷酸化和钙的调节，结合cPLA2α的C2结构域，并诱导其转位到高尔基体、内质网和核膜。这项建议的具体目的是确定C2结构域和催化结构域中的残基，这些残基决定了膜的靶向性和与膜的稳定结合。还将确定cPLA2α的脂结合特异性，并确定C2结构域和催化结构域中与阴离子磷脂相互作用的残基。钙和磷酸化在调节(1)通过不同信号通路的激动剂在CHO细胞中的移位中的作用，以及(2)cPLA2α与从CHO细胞分离的高尔基体膜的相互作用将被研究。在cPLA2α/-小鼠肺成纤维细胞中发现了另一类PLA2，即cPLA2β，它为花生四烯酸的释放和PGE2的产生提供了另一种途径。我们将研究cPLA2β的酶学性质及其C2结构域的特征。这些结果将提供有关cPLA2α调控的详细信息，cPLA2α是二十烷类化合物生产中的第一个关键调节酶，并提供有关cPLA2β性质的新信息。

DESCRIPTION (provided by applicant): Group IVA cytosolic phospholipase A2alpha (cPLA2alpha) mediates agonist-induced release of arachidonic acid, the precursor for eicosanoids. These potent lipid mediators, which include prostaglandins and leukotrienes, are required for many normal physiological processes and also promote acute inflammation. Eicosanoids produced as a result of cPLA2alpha activation have been implicated in the development of acute lung injury and pulmonary fibrosis. The objectives of this proposal are to elucidate the mechanisms involved in targeting cPLA2alpha to membrane and regulation of its hydrolytic activity, cPLA2alpha is regulated by phosphorylation and calcium, which binds to the C2 domain of cPLA2alpha and induces its translocation to Golgi, ER and nuclear membrane. The specific aims of this proposal are to identify residues in the C2 domain and catalytic domain that determine membrane targeting and stable association with membrane. The lipid binding specificity of cPLA2alpha will also be determined, and residues in the C2 domain and catalytic domain that interact with anionic phospholipids identified. The role of calcium and phosphorylation in regulating (1) translocation of cPLA2alpha in CHO cells by agonists that act through distinct signaling pathways, and (2) the interaction of cPLA2alpha with Golgi membranes isolated from CHO cells will be studied. Another Group IV PLA2, cPLA2beta was identified in lung fibroblasts from cPLA2alpha-/- mice, and it provided an alternative pathway for arachidonic acid release and PGE 2 production. The enzymatic properties of cPLA2beta and characteristics of its C2 domain will be studied. The results will provide detailed information about the regulation of cPLA2alpha, the first key regulatory enzyme in eicosanoid production, and new information about the properties of cPLA2beta.