Approaches for investigating the extracellular signaling function of ISG15.

ABSTRACT: ISG15 is a ubiquitin-like protein (Ubl) that is expressed in response to Type 1 Interferon (IFN-?/?) signaling. Remarkably, ISG15 has three distinct biochemical activities involved in innate immune responses to viral and/or microbial infections. The canonical function of ISG15 is as a posttranslational modifier, and protein ISGylation has been demonstrated to be antiviral. A second intracellular function, independent of conjugation activity, is attenuation of IFN-?/? signaling at the interferon receptor, which appears to be important for terminating IFN responses. The third function of ISG15, and the focus of this chapter, is as an extracellular signaling molecule that promotes the secretion of Type 2 Interferon (IFN-?) by Natural Killer (NK) cells. This function is important for control of microbial infections, including mycobacterial infections. Here, we describe methods for purification of ISG15, preparation, and culture of primary peripheral blood mononuclear cells (PBMCs) and NK-92 cells, assays for IL-12- and ISG15-dependent cytokine (IFN-? and IL-10) secretion, and assays for initial intracellular signaling events triggered by extracellular ISG15.

SUBMITTER: Swaim CD 

PROVIDER: S-EPMC6472260 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature