Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation
Crucial role of two potential cytosolic regions of Nox2, 191TSSTKTIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidase activation
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DOI：
10.1074/jbc.m500226200
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发表时间：
2005-04-15
影响因子：
4.8
通讯作者：
Stasia, MJ
中科院分区：
文献类型：
--
作者：
Li, XJ;Grunwald, D;Stasia, MJ
细胞溶质因子p67（phox）和p47（phox）与细胞色素B（558）的组装是NADPH氧化酶激活的关键之一。Nox 2的某些序列似乎参与胞质因子相互作用。研究了Nox 2的D-环（TSSTKTIRRS 200）-T-191和C-末端（484）DESQANHFAVHHDEEKD（500）对氧化酶活性和组装的作用。通过定向诱变将带电荷的氨基酸突变为中性或反向电荷以产生21个突变体。在X-CGD PLB-985细胞模型中表达重组野生型或突变型Nox 2。Nox 2基因D环中的K195 A/E、R198 E、R199 E和RR 198 199 QQ/AA突变完全消除了氧化酶活性。然而，这些D-环突变体表现出正常的p47（phox）易位和碘硝基四唑（INT）还原酶活性，这表明该区域的带电氨基酸是从FAD到氧的电子转移所必需的。用Nox 1、Nox 3或Nox 4的同源物替换Nox 2 D环是完全功能性的。此外，fMLP（甲酰甲硫氨酰亮氨酰苯丙氨酸）激活的R199 Q-Nox 2和D-环（Nox 4）-Nox 2突变体表现出的NADPH氧化酶活性是对照细胞的4至8倍，表明这些突变导致更有效的氧化酶激活过程。相反，Nox 2 α-螺旋环的D484 T和D500 A/R/G突变体没有表现出与缺陷性p47（phox）膜转位相关的NADPH氧化酶和INT还原酶活性。这表明Nox 2的C-末端的α-螺旋环可能参与激活期间发生的NADPH氧化酶复合物的正确组装，允许胞质因子易位和电子从NADPH转移到FAD。
Assembly of cytosolic factors p67(phox) and p47(phox) with cytochrome b(558) is one of the crucial keys for NADPH oxidase activation. Certain sequences of Nox2 appear to be involved in cytosolic factor interaction. The role of the D-loop (TSSTKTIRRS200)-T-191 and the C-terminal (484)DESQANHFAVHHDEEKD(500) of Nox2 on oxidase activity and assembly was investigated. Charged amino acids were mutated to neutral or reverse charge by directed mutagenesis to generate 21 mutants. Recombinant wild-type or mutant Nox2 were expressed in the X-CGD PLB-985 cell model. K195A/E, R198E, R199E, and RR198199QQ/AA mutations in the D-loop of Nox2 totally abolished oxidase activity. However, these D-loop mutants demonstrated normal p47(phox) translocation and iodonitrotetrazolium (INT) reductase activity, suggesting that charged amino acids of this region are essential for electron transfer from FAD to oxygen. Replacement of Nox2 D-loop with its homolog of Nox1, Nox3, or Nox4 was fully functional. In addition, fMLP (formylmethionylleucylphenylalanine)activated R199Q-Nox2 and D-loop(Nox4)-Nox2 mutants exhibited four to eight times the NADPH oxidase activity of control cells, suggesting that these mutations lead to a more efficient oxidase activation process. In contrast, the D484T and D500A/R/G mutants of the alpha-helical loop of Nox2 exhibited no NADPH oxidase and INT reductase activities associated with a defective p47(phox) membrane translocation. This suggests that the alpha-helical loop of the C-terminal of Nox2 is probably involved in the correct assembly of the NADPH oxidase complex occurring during activation, permitting cytosolic factor translocation and electron transfer from NADPH to FAD.