Fungal metabolite gliotoxin targets flavocytochrome b558 in the activation of the human neutrophil NADPH oxidase
Fungal metabolite gliotoxin targets flavocytochrome b558 in the activation of the human neutrophil NADPH oxidase
复制标题
DOI：
10.1128/iai.73.1.235-244.2005
复制
发表时间：
2005-01-01
影响因子：
3.1
通讯作者：
Tsunawaki, S
中科院分区：
文献类型：
--
作者：
Nishida, S;Yoshida, LS;Tsunawaki, S
真菌胶质毒素（GT）是中性粒细胞产生O-2（-）的NADPH氧化酶的有效抑制剂。我们报道了GT处理的中性粒细胞不能磷酸化p47(phox)，这是酶激活的必要步骤，因为GT阻止了蛋白激酶C β i与p47（phox）在膜上的共定位。然而，GT是否直接影响NADPH氧化酶组分仍未得到解答。在这里，我们在无细胞激活实验中研究了GT对NADPH氧化酶成分的影响。当使用两种不同的电子受体进行检测时，经GT处理的中性粒细胞膜产生O-2（-）的能力分别比未处理的中性粒细胞低40.0%和30.6%，这表明黄细胞色素b（558）在细胞中受到了GT的影响。相反，相应的细胞质仍能被激活。接下来，将GT添加到由黄细胞色素b（558）和细胞质成分(天然细胞质或重组p67（phox), p47（phox）和Rac2）组成的实验中，在用肉豆蔻酸刺激之前进行，会产生显著的抑制作用（50%抑制浓度= 3.3 muM）。GT也能阻止NADPH的消耗，但p67（phox）、p47（phox）和Rac2在体外与黄细胞色素b（558）的组装是正常的。后向活性酶中添加GT是无效的。用GT单独处理膜也会导致黄细胞色素b（558）重建O-2（-）生成的能力明显丧失，在细胞水平上支持了这一结论。然而，经gt处理的膜的黄细胞色素b（558）血红素光谱保持不变，表明血红素保持完整。这些结果表明，GT直接损害了黄细胞色素b（558）中电子传递的关键位点，这些位点只有在氧化酶激活之前才能进入。
Fungal gliotoxin (GT) is a potent inhibitor of the O-2(-)-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47(phox), a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C betaII with p47(phox) on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O-2(-)-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b(558) is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b(558) and cytosolic components (native cytosol or recombinant p67(phox), p47(phox), and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 muM) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67(phox), p47(phox), and Rac2 with flavocytochrome b(558) is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b(558)'s ability to reconstitute O-2(-) generation, supporting the conclusion at the cellular level. The flavocytochrome b(558) heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b(558), which is accessible only before oxidase activation.