A Proteomic Investigation Characterizing the Interactions and Posttranslational Modifications of the Interferon-Inducible Protein IFI16

Abstract

The mammalian immune system has evolved multiple methods of combating pathogens, with the detection of pathogenic nucleic acids being one of the first lines of defense. Interferon-inducible protein IFI16 has become a focus of research in recent years as a sensor of pathogenic DNA that is able to stimulate both a type I IFN response and a proinflammatory response. Although it is mostly found in the nucleus, IFI16 has been shown to sense DNA in the cytoplasm as well. Despite these findings, much about the mechanisms regulating its ability to sense foreign DNA and distinguish it from host DNA remains uncharacterized. In this study, a mass spectrometry-based approach was used to gain a better understanding of IFI16’s function and role in innate immunity. IFI16 was isolated in the nuclear and cytoplasmic fractions of differentiated THP-1 monocytes, immunoaffinity purified and analyzed by tandem mass spectrometry in order to determine its protein-protein interactions and posttranslational modifications. Here, it is shown that the interactions of IFI16 in THP-1 cells differ from those of HEK293, which is important given its function in the innate immune system. Additionally, IFI16 was shown to interact with the cytoplasmic nucleic acid sensor LRRFIP1 and a repressing factor of NF-κB (NKRF) in the nucleus. HERC5, an E3 ligase essential for the conjugation of ISG15 was shown to specifically interact with IFI16 after IFN induction. Finally, differential acetylations and phosphorylations were identified in the cytoplasmic and nuclear fractions of THP-1 cells. Taken together, these data suggest that IFI16’s interacting partners are cell-specific and localization-dependent and the same can be said of its posttranslational modifications. Moreover, IFI16’s interactions are different in IFNinduced cells, which speaks to the versatility of this pathogenic DNA sensor.