Time-resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes
Time-resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes
复制标题
DOI：
10.1096/fj.15-278010
复制
发表时间：
2016-01-01
期刊：
影响因子：
4.8
通讯作者：
Garscha, Ulrike
中科院分区：
文献类型：
--
作者：
Gerstmeier, Jana;Weinigel, Christina;Garscha, Ulrike
5-脂氧合酶（5-LO）催化促炎性白三烯生物合成的初始步骤。在细胞活化时，5-LO易位至核膜，在核膜处花生四烯酸通过5-LO活化蛋白（FLAP）转移至5-LO进行代谢。虽然先前的数据表明5-LO与FLAP的关联，但是天然5-LO/FLAP复合物的原位组装仍然是难以捉摸的。在这里，我们显示时间分辨5-LO/FLAP共定位的免疫荧光显微镜和原位5-LO/FLAP相互作用的邻近连接试验在核膜的Ca 2 +-ionophoreA 23187激活的人单核细胞和中性粒细胞的5-LO活性。虽然5-LO易位和产品的形成是在1.5-3分钟内完成，5-LO/FLAP相互作用被延迟，并进行长达30分钟。虽然单核细胞和中性粒细胞含有相当数量的5-LO蛋白，中性粒细胞产生3-5倍高水平的5-LO产品由于长期的活动，伴随着延迟5-LO核膜易位。花生四烯酸似乎充当5-LO/FLAP组装的接头，而FLAP抑制剂（MK 886，100 nM; BAY X 1005，3 μ M）破坏复合物。我们的结论是，FLAP可以调节5-LO活性在2种方式：首先通过诱导一个初始的灵活的协会有效的5-LO产品的合成，然后通过形成一个紧密的5-LO/FLAP复合物，终止5-LO活动。
5-Lipoxygenase (5-LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5-LO translocates to the nuclear membrane where arachidonic acid is transferred by 5-LO-activating protein (FLAP) to 5-LO for metabolism. Although previous data indicate association of 5-LO with FLAP, the in situ assembly of native 5-LO/FLAP complexes remains elusive. Here, we show time-resolved 5-LO/FLAP colocalization by immunofluorescence microscopy and in situ 5-LO/FLAP interaction by proximity ligation assay at the nuclearmembrane of Ca2+-ionophoreA23187-activated human monocytes and neutrophils in relation to 5-LO activity. Although 5-LO translocation and product formation is completed within 1.5-3 min, 5-LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5-LO protein, neutrophils produce 3-5 times higher levels of 5-LO products due to prolonged activity, accompanied by delayed 5-LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5-LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 mu M) disrupt the complex. We conclude that FLAP may regulate 5-LO activity in 2 ways: first by inducing an initial flexible association for efficient 5-LO product synthesis, followed by the formation of a tight 5-LO/FLAP complex that terminates 5-LO activity.