Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity
Peroxiredoxin 6 translocates to the plasma membrane during neutrophil activation and is required for optimal NADPH oxidase activity
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DOI：
10.1016/j.bbamcr.2011.11.014
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发表时间：
2012-02-01
影响因子：
5.1
通讯作者：
Leto, Thomas L.
中科院分区：
文献类型：
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作者：
Ambruso, Daniel R.;Ellison, Michael A.;Leto, Thomas L.
中性粒细胞部分通过产生活性氧物质（ROS）提供抵抗微生物入侵的第一道防线，活性氧物质（ROS）通过激活烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶产生超氧阴离子（O-2（-））介导。吞噬细胞氧化酶（phox）有多种蛋白质成分，在刺激的中性粒细胞质膜上组装。我们最近描述了中性粒细胞中的一种蛋白质，过氧化物酶6（Prdx 6），它具有过氧化物酶和磷脂酶A2（PLA 2）的活性，并在SDS激活的无细胞系统中增强氧化酶活性。进一步研究了Prdx 6在phox活性中的作用。在重组的phox感受态K562细胞中，siRNA介导的Prdx 6抑制导致响应于甲酰-甲硫氨酰-亮氨酰-苯丙氨酸（fMLP）或佛波醇肉豆蔻酸酯（PMA）的NADPH氧化酶活性降低。在PMA刺激的中性粒细胞中，Prdx 6易位到质膜，如Western印迹和共聚焦显微镜所示。Prdx 6在phox感受态K562细胞中的易位需要p67 phox和p47 phox。此外，PMA刺激的，氧化酶主管K562细胞与siRNA介导的Prdx 6抑制的质膜含有较少的p47 phox和p67 phox相比，其中Prdx 6没有减少的细胞。无细胞氧化酶试验表明，重组Prdx 6没有改变NADPH的Km，但增加了O-2（-）生产的V-max在一个饱和，Prdx 6浓度依赖性的方式。在Prdx（C47 S）和磷脂酶（S32 A）活性中具有突变的重组蛋白质都将无细胞phox活性增强到与野生型蛋白质相同的程度。Prdx 6支持活性氧化酶复合物保留在受刺激的质膜中，突变蛋白的结果意味着Prdx 6在支持最佳NADPH氧化酶活性方面发挥额外的生化或结构作用。(C)2011 Elsevier B. V.保留所有权利。
Neutrophils provide the first line of defense against microbial invasion in part through production of reactive oxygen species (ROS) which is mediated through activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generating superoxide anion (O-2(-)). The phagocyte oxidase (phox) has multiple protein components that assemble on the plasma membrane in stimulated neutrophils. We recently described a protein in neutrophils, peroxiredoxin 6 (Prdx6), which has both peroxidase and phospholipase A2 (PLA2) activities and enhances oxidase activity in an SDS-activated, cell-free system. The function of Prdx6 in phox activity is further investigated. In reconstituted phox-competent K562 cells, siRNA-mediated suppression of Prdx6 resulted in decreased NADPH oxidase activity in response to formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA). In neutrophils stimulated with PMA, Prdx6 translocated to plasma membrane as demonstrated by Western blot and confocal microscopy. Translocation of Prdx6 in phox competent K562 cells required both p67phox and p47phox. In addition, plasma membrane from PMA-stimulated, oxidase competent K562 cells with siRNA-mediated Prdx6 suppression contained less p47phox and p67phox compared to cells in which Prdx6 was not decreased. Cell-free oxidase assays showed that recombinant Prdx6 did not alter the K-m for NADPH, but increased the V-max for O-2(-) production in a saturable, Prdx6 concentration-dependent manner. Recombinant proteins with mutations in Prdx (C47S) and phospholipase (S32A) activity both enhanced cell-free phox activity to the same extent as wild type protein. Prdx6 supports retention of the active oxidase complex in stimulated plasma membrane, and results with mutant proteins imply that Prdx6 serves an additional biochemical or structural role in supporting optimal NADPH oxidase activity. (C) 2011 Elsevier B.V. All rights reserved.