Direct interaction of the novel nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase
Direct interaction of the novel nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase
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DOI：
10.1074/jbc.m406486200
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发表时间：
2004-10-29
影响因子：
4.8
通讯作者：
Brandes, RP
中科院分区：
文献类型：
--
作者：
Ambasta, RK;Kumar, P;Brandes, RP
Nox 1和Nox 4是白细胞NADPH氧化酶亚基Nox 2（gp91phox）的同源物，在各种细胞类型中介导超氧阴离子的形成。然而，它们与NADPH氧化酶的其他组分的相互作用很难确定。我们确定了Nox1和Nox4与NADPH氧化酶的p22phox亚基是否发生直接相互作用。利用共聚焦显微镜，共定位的p22 phox与Nox 1，Nox 2，和Nox 4的瞬时转染血管平滑肌细胞（VSMC）和HEK 293细胞中观察到。构建了编码p22phox和Nox蛋白与青色和黄色荧光蛋白（分别为cfp和yfp）的荧光融合蛋白的质粒，并在VSMC和HEK293细胞中表达。与Nox 1或Nox 4共转染后，cfp标记的p22 phox表达水平增加。利用荧光共振能量转移技术观察了p22phox与Nox荧光融合蛋白之间的相互作用。天然Nox 1从人VSMC的免疫沉淀显示共沉淀的p22phox。从转染的HEK293细胞的免疫沉淀揭示了天然p22phox与yfp标记的Nox1，Nox2和Nox4的共沉淀。在组氨酸（对应于人Nox 2中的位置115）突变为亮氨酸之后，这种相互作用被消除。转染大鼠p22 phox（而不是Noxo1和Noxa1）增加了表达Nox4的细胞中的自由基生成。我们提供的证据表明，p22phox直接与Nox1和Nox4相互作用，形成一个产生超氧化物的NADPH氧化酶，并证明在Nox蛋白的潜在血红素结合位点的突变破坏了Nox1和Nox4与p22phox的复合物形成。
Nox1 and Nox4, homologues of the leukocyte NADPH oxidase subunit Nox2 (gp91phox) mediate superoxide anion formation in various cell types. However, their interactions with other components of the NADPH oxidase are poorly defined. We determined whether a direct interaction of Nox1 and Nox4 with the p22phox subunit of the NADPH oxidase occurs. Using confocal microscopy, co-localization of p22phox with Nox1, Nox2, and Nox4 was observed in transiently transfected vascular smooth muscle cells (VSMC) and HEK293 cells. Plasmids coding for fluorescent fusion proteins of p22phox and the Nox proteins with cyan- and yellow-fluorescent protein (cfp and yfp, respectively) were constructed and expressed in VSMC and HEK293 cells. The cfp-tagged p22phox expression level increased upon co-transfection with Nox1 or Nox4. Protein-protein interaction between the fluorescent fusion proteins of p22phox and the Nox partners was observed using the fluorescence resonance energy transfer technique. Immunoprecipitation of native Nox1 from human VSMC revealed co-precipitation of p22phox. Immunoprecipitation from transfected HEK293 cells revealed co-precipitation of native p22phox with yfp-tagged Nox1, Nox2, and Nox4. Following mutation of a histidine (corresponding to the position 115 in human Nox2) to leucine, this interaction was abolished. Transfection of rat p22phox ( but not Noxo1 and Noxa1) increased the radical generation in cells expressing Nox4. We provide evidence that p22phox directly interacts with Nox1 and Nox4, to form an superoxide-generating NADPH oxidase and demonstrate that mutation of the potential heme binding site in the Nox proteins disrupts the complex formation of Nox1 and Nox4 with p22phox.