Helmcke Ina, Heumüller Sabine, Tikkanen Ritva, Schröder Katrin, Brandes Ralf P
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Frankfurt am Main, Germany.
Antioxid Redox Signal. 2009 Jun;11(6):1279-87. doi: 10.1089/ars.2008.2383.
Nox NADPH oxidases differ in their mode of activation, subcellular localization, and physiological function. Nox1 releases superoxide anions (O(2)(-)) and depends on cytosolic activator proteins, whereas Nox4 extracellularly releases hydrogen peroxide (H(2)O(2)), and its activity does not require cotransfection of additional proteins. We constructed chimeric proteins consisting of Nox1 and Nox4 expressed in HEK293 cells. When the cytosolic tail of Nox4 was fused with the transmembrane part of Nox1, Nox1 became constitutively active. The reciprocal construct was inactive, suggesting that cytosolic subunit-dependent activation requires elements in the transmembrane loops. By TIRF-microscopy, Nox1 was observed in the plasma membrane, whereas Nox4 colocalized with proteins of the endoplasmic reticulum. Fusion proteins of Myc and Nox revealed that the N-terminal part of Nox1 but not Nox4 is cleaved. When the potential signal peptide of Nox4 was inserted into Nox1, plasma-membrane localization was lost, and the protein was retained in vesicle-like structures below the plasma membrane. The potential signal peptide of Nox1 failed to translocate Nox4 to the plasma membrane but switched the extracellularly detectable ROS from H(2)O(2) to O(2)(-). Thus, the very N-terminal part of Nox proteins determines subcellular localization and the ROS type released, whereas the cytosolic tail regulates activity.
Nox 烟酰胺腺嘌呤二核苷酸磷酸氧化酶在激活方式、亚细胞定位和生理功能上存在差异。Nox1 释放超氧阴离子（O₂⁻），并依赖于胞质激活蛋白，而 Nox4 在细胞外释放过氧化氢（H₂O₂），其活性不需要共转染其他蛋白质。我们构建了由 Nox1 和 Nox4 组成的嵌合蛋白，并在人胚肾 293 细胞（HEK293）中表达。当 Nox4 的胞质尾部与 Nox1 的跨膜部分融合时，Nox1 变得组成型激活。反向构建体无活性，这表明依赖胞质亚基的激活需要跨膜环中的元件。通过全内反射荧光显微镜（TIRF-显微镜）观察到，Nox1 位于质膜中，而 Nox4 与内质网蛋白共定位。Myc 和 Nox 的融合蛋白显示，Nox1 的 N 端部分而非 Nox4 的 N 端部分被切割。当将 Nox4 的潜在信号肽插入 Nox1 时，质膜定位丧失，该蛋白保留在质膜下方的囊泡样结构中。Nox1 的潜在信号肽未能将 Nox4 转运到质膜，但将细胞外可检测到的活性氧从 H₂O₂ 转换为 O₂⁻。因此，Nox 蛋白的最 N 端部分决定亚细胞定位和释放的活性氧类型，而胞质尾部调节活性。
