A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia.
A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia.
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DOI：
10.1007/s12035-020-02042-w
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发表时间：
2020-11
影响因子：
5.1
通讯作者：
Guilarte TR
中科院分区：
文献类型：
--
作者：
Loth MK;Guariglia SR;Re DB;Perez J;de Paiva VN;Dziedzic JL;Chambers JW;Azzam DJ;Guilarte TR
在脑神经元中，转运蛋白18 kDa（TSPO）是一种应激反应蛋白，在不同的中枢神经系统病理中的小胶质细胞和星形胶质细胞中上调。TSPO被广泛用作临床前和临床神经影像学研究中神经炎症的生物标志物。然而，对TSPO在神经胶质细胞中的功能了解甚少。在这项研究中，我们探讨了TSPO和NADPH氧化酶2（NOX 2）在小胶质细胞之间的相互作用。我们发现TSPO与gp 91 phox和p22 phox相关，这是原代小鼠小胶质细胞中NOX 2的主要亚基。采用免疫共沉淀、共聚焦免疫荧光成像和邻位连接试验观察TSPO与gp 91 phox和p22 phox的结合。我们发现，除了gp 91 phox和p22 phox，电压依赖性阴离子通道（VDAC）也与TSPO免疫共沉淀与以前的报告一致。当我们比较脂多糖（LPS）刺激的小胶质细胞与载体对照时，我们发现较低量的gp 91 phox和p22 phox蛋白与TSPO共免疫沉淀，表明TSPO-NOX 2亚基缔合的破坏。TSPO免疫金电子显微镜证实TSPO存在于线粒体外膜中，但它也存在于内质网（ER）、内质网相关的ER膜（MAM）和质膜中。TSPO在MAM的定位可能代表了TSPO与gp 91 phox和p22 phox相互作用的亚细胞位点，因为MAM是线粒体外膜蛋白（TSPO）和ER蛋白（gp 91 phox和p22 phox）之间的通讯点，在此它们成熟并形成细胞色素b558（Cytb 558）异二聚体。我们还发现，活性氧（ROS）的急性爆发增加了小胶质细胞表面上的TSPO水平，这种作用被ROS清除剂消除。这些结果表明，ROS的产生可能会改变TSPO的亚细胞分布。总的来说，我们的研究结果表明，在小胶质细胞中，TSPO与主要的NOX 2亚基gp 91 phox和p22 phox相关。我们推测这种相互作用可能调节Cytb 558的形成，并调节小胶质细胞中的NOX 2水平、ROS产生和氧化还原稳态。本文的在线版本（10.1007/s12035-020-02042-w）包含补充材料，可供授权用户使用。
In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91phox and p22phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) where they mature and form the cytochrome b558 (Cytb558) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia. The online version of this article (10.1007/s12035-020-02042-w) contains supplementary material, which is available to authorized users.
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影响因子：
4.1
作者：
Batarseh, Amani;Papadopoulos, Vassilios
通讯作者：
Papadopoulos, Vassilios
影响因子：
9
作者：
Gatliff J;East DA;Singh A;Alvarez MS;Frison M;Matic I;Ferraina C;Sampson N;Turkheimer F;Campanella M
通讯作者：
Campanella M
影响因子：
6.2
作者：
Choi, Judy;Ifuku, Masataka;Noda, Mami;Guilarte, Tomas R.
通讯作者：
Guilarte, Tomas R.
影响因子：
3.4
作者：
ASAGAMI, H;HINO, Y;TAKESHIGE, K
通讯作者：
TAKESHIGE, K
DOI：
10.1016/j.tem.2015.04.001
发表时间：
2015-07
期刊：
Trends in endocrinology and metabolism: TEM
影响因子：
--
作者：
Gut P;Zweckstetter M;Banati RB
通讯作者：
Banati RB