Microglia are myeloid cells in the central nervous system which maintain homeostasis and contribute to repair, but instigate neuroinflammation when are activated by infection, trauma or neurological diseases. Initiation of acute inflammatory responses could be mimicked in vitro by stimulation of microglial cultures with lipopolysaccharide (LPS). We have previously demonstrated Stat-dependent induction of the Uba7 mRNA expression in LPS stimulated microglia. Uba7 is an E1 enzyme crucial for posttranslational protein modifications. ISG'ylation is a process in which ISG15 is covalently attached to lysines of target proteins via the sequential action of three enzymes: the E1-activating enzyme UbE1L (UBA7), the E2-conjugating enzyme UBCH8, and E3 ligase HERC5. Here we use quantitative labeled-free mass spectrometry and gene silencing to determine the role of ISG'ylation in LPS-stimulated microglia. We found the increased mRNA levels of Isg15, Uba7, Ube2l6, Herc6 and profound ISG'ylation in inflammatory microglia. Silencing of Uba7 in BV2 microglial cells results in a profound decrease in the level of hundreds proteins as measured by mass spectrometry. There is statistically significant intersection of Uba7-dependent proteins in LPS-stimulated microglia and three datasets of ISG'ylated proteins reported in earlier studies. Stat1, a main activator of Uba7 expression, was modified by ISG15 after LPS stimulation. The level of both total and phospho-Stat1 is decreased after Uba7 knockdown leading to premature termination of immune responses as evidenced by the reduction of iNos and Ccl5 expression. Our results suggest that increased ISG'ylation in LPS-stimulated microglia supports stability of proteins, including Stat1, which prevents termination of immune responses during inflammation.

小胶质细胞是中枢神经系统中的髓样细胞，它们维持体内稳态并有助于修复，但是当被感染，创伤或神经系统疾病激活时会诱发神经炎症。急性炎症反应的引发可以模仿在体外用脂多糖（LPS）小胶质细胞培养的刺激。我们之前已经证明了Uba7的Stat依赖性诱导LPS刺激的小胶质细胞中的mRNA表达。Uba7是一种E1酶，对翻译后蛋白质修饰至关重要。ISG酰化是一个过程，其中ISG15通过三种酶（E1激活酶UbE1L（UBA7），E2缀合酶UBCH8和E3连接酶HERC5）的顺序作用共价连接到目标蛋白的赖氨酸上。在这里，我们使用定量无标记质谱和基因沉默来确定ISG'ylation在LPS刺激的小胶质细胞中的作用。我们发现Isg15，Uba7，Ube2l6，Herc6的mRNA水平升高和炎症性小胶质细胞中的深层ISG'ylation。通过质谱法测定，BV2小胶质细胞中Uba7的沉默导致数百种蛋白质水平的显着降低。在LPS刺激的小胶质细胞中，UBa7依赖性蛋白与早期研究中报道的三个ISG'酰化蛋白数据集之间存在统计学上的显着交叉。Stat1是Uba7表达的主要激活因子，在LPS刺激后被ISG15修饰。Uba7敲低导致总免疫和磷酸化Stat1的水平降低，从而导致免疫反应的提前终止，这可通过iNos和Ccl5的降低来证明表达。我们的结果表明，LPS刺激的小胶质细胞中ISG'ylation的增加支持蛋白质（包括Stat1）的稳定性，从而阻止炎症过程中免疫反应的终止。