Institut de Chimie Physique, UMR8000, Université Paris-Saclay, CNRS, 91405 Orsay, France.
Institut de Chimie Physique, UMR8000, Université Paris-Saclay, CNRS, 91405 Orsay, France.
Biochim Biophys Acta Mol Cell Res. 2022 Sep;1869(9):119276. doi: 10.1016/j.bbamcr.2022.119276. Epub 2022 Apr 27.
The phagocyte NADPH oxidase (NOX2) is a key enzyme of the innate immune system generating superoxide anions (O), precursors of reactive oxygen species. The NOX2 protein complex is composed of six subunits: two membrane proteins (gp91 and p22) forming the catalytic core, three cytosolic proteins (p67, p47 and p40) 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 p47 (aa 1-286), p67 (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.
吞噬细胞 NADPH 氧化酶（NOX2）是先天免疫系统的关键酶，可生成超氧阴离子（O），这是活性氧物质的前体。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（gp91 和 p22），三个胞质蛋白（p67、p47 和 p40）和一个小 GTP 酶 Rac。NADPH 氧化酶的复杂激活机制依赖于将胞质亚基与膜结合成分组装在一起。一种称为“Trimera”的嵌合蛋白，由胞质蛋白 p47（aa1-286）、p67（aa1-212）和全长 Rac1Q61L 的必需结构域组成，可在无需任何刺激的情况下使细胞中的 NOX2 活性持续且强烈。我们将 Trimera 用作活细胞中吞噬细胞 NADPH 氧化酶的单一激活蛋白，并研究了这种持续且长期的 NOX 活性对细胞生理学的影响。我们发现，持续的高水平 NOX 活性会导致细胞内 pH 值酸化，引发细胞凋亡，并导致细胞膜中脂质的局部过氧化。这些局部的膜损伤与通过 FRET-FLIM 显微镜观察到的 Trimera 在质膜中强烈聚集的趋势相关。
