The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O2·-), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91phox and p22phox) forming the catalytic core, three cytosolic proteins (p67phox, p47phox and p40phox) and a small GTPase Rac. The sophisticated activation mechanism of the NADPH oxidase relies on the assembly of cytosolic subunits with the membrane-bound components. A chimeric protein, called Trimera, composed of the essential domains of the cytosolic proteins p47phox (aa 1-286), p67phox (aa 1-212) and full-length Rac1Q61L, enables a constitutive and robust NOX2 activity in cells without the need of any stimulus. We employed Trimera as a single activating protein of the phagocyte NADPH oxidase in living cells and examined the consequences on the cell physiology of this continuous and long-term NOX activity. We showed that the sustained high level of NOX activity causes acidification of the intracellular pH, triggers apoptosis and leads to local peroxidation of lipids in the membrane. These local damages to the membrane correlate with the strong tendency of the Trimera to clusterize in the plasma membrane observed by FRET-FLIM microscopy.

中文翻译：

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活细胞中本构NOX2活性的后果：胞浆酸化，凋亡和局部脂质过氧化

吞噬细胞NADPH氧化酶（NOX2）是先天免疫系统的关键酶，可产生超氧阴离子（O2·-），这是活性氧的前体。NOX2蛋白复合物由六个亚基组成：两个形成催化核心的膜蛋白（gp91phox和p22phox），三个胞质蛋白（p67phox，p47phox和p40phox）和一个小的GTPase Rac。NADPH氧化酶的复杂激活机制取决于具有膜结合成分的胞质亚基的组装。一种嵌合蛋白，称为Trimera，由胞质蛋白p47phox（aa 1-286），p67phox（aa 1-212）和全长Rac1Q61L的基本域组成，可在细胞中实现组成型且稳定的NOX2活性，而无需任何刺激。我们将Trimera用作吞噬细胞NADPH氧化酶在活细胞中的单一激活蛋白，并研究了这种连续和长期NOX活性对细胞生理的影响。我们表明，持续高水平的NOX活性会引起细胞内pH的酸化，触发细胞凋亡并导致膜中脂质的局部过氧化。通过FRET-FLIM显微镜观察到，这些对膜的局部损伤与Trimera在质膜中聚集的强烈趋势相关。