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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.Uba7 plays a crucial role in conjugating ISG15 to other proteins (ISG'ylation).ISG'ylation increases stability of targeted proteins in inflammatory microglia.Uba7 is a positive regulator of Stat1-driven gene transcription.Uba7 and ISG'ylation prevent premature termination of immune responses.