Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways

摘要

IFN-α/β plays an essential role in innate immunity against viral and bacterial infection. Among the proteins induced by IFN-α/β are the ubiquitin-like ISG15 protein and its E1- (Ube1L) and E2- (UbcH8) conjugating enzymes, leading to the conjugation of ISG15 to cellular proteins. It is likely that ISG15 conjugation plays an important role in antiviral response because a human virus, influenza B virus, inhibits ISG15 conjugation. However, the biological function of ISG15 modification remains unknown, largely because only a few human ISG15 target proteins have been identified. Here we purify ISG15-modified proteins from IFN-β-treated human (HeLa) cells by using double-affinity selection and use mass spectroscopy to identify a large number (158) of ISG15 target proteins. Eight of these proteins were subjected to further analysis and verified to be ISG15 modified in IFN-β-treated cells, increasing the likelihood that most, if not all, targets identified by mass spectroscopy are bona fide ISG15 targets. Several of the targets are IFN-α/β-induced antiviral proteins, including PKR, MxA, HuP56, and RIG-I, providing a rationale for the inhibition of ISG15 conjugation by influenza B virus. Most targets are constitutively expressed proteins that function in diverse cellular pathways, including RNA splicing, chromatin remodeling polymerase II transcription, cytoskeleton organization and regulation, stress responses, and translation. These results indicate that ISG15 conjugation impacts nuclear as well as cytoplasmic functions. By targeting a wide array of constitutively expressed proteins, ISG15 conjugation greatly extends the repertoire of cellular functions that are affected by IFN-α/β.