5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation.
5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation.
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DOI：
10.1172/jci117889
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发表时间：
1995-05
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影响因子：
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通讯作者：
John W Woods;Michael J. Coffey;T. Brock;Irwin I. Singer;Marc Peters-Golden
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文献类型：
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作者：
John W Woods;Michael J. Coffey;T. Brock;Irwin I. Singer;Marc Peters-Golden
5-脂氧合酶（5-LO）和5-脂氧合酶激活蛋白（FLAP）是花生四烯酸合成白三烯（LT）的两个关键蛋白。尽管肺泡巨噬细胞（AM）和外周血白细胞（PBL）在激活后都会产生大量的LT，但在PBL激活后，5-LO从可溶性池转移到颗粒部分，但无论是否激活，5-LO都包含在AM的颗粒部分中。因此，我们检查了从正常供体收集的自体人AM和PBL中5-LO的亚细胞定位。免疫金电镜显示静止PBL中几乎没有5-LO，而静止AM在细胞核的常染色质区域显示出丰富的5-LO表位。通过细胞分离和免疫印迹分析以及间接免疫荧光显微镜证实静息AM细胞核中存在大量5-LO。在A23187激活的AM和PBL中，所有可观察到的5-LO免疫金标记都与核膜相关。在这两种类型的静息细胞中，FLAP主要与核膜相关，其定位不受A23187激活的影响。结合FLAP的MK-886在离子载体刺激的AM和PBL中检测了其作用。虽然MK-886抑制两种细胞类型的LT合成，但它不能阻止5-LO向核膜的易位。这些结果表明，在活化AM和PBL中，核膜是5-LO与FLAP和花生四烯酸相互作用以催化LT合成的位点，而在静止AM中，核的常染色质区域是易位酶的主要来源。此外，LT合成是一个两步过程，包括不依赖于flap的5-LO向核膜的易位，然后是依赖于flap的酶的激活。
5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.