Human ISG15 conjugation targets both IFN-induced and constitutively expressed proteins functioning in diverse cellular pathways.
IFN-alpha/beta plays an essential role in innate immunity against viral and bacterial infection. Among the proteins induced by IFN-alpha/beta 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-beta-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-beta-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-alpha/beta-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-alpha/beta.
Zhao
Chen
C
Institute for Cellular and Molecular Biology, University of Texas, 2500 Speedway, Austin, TX 78712, USA.
Denison
Carilee
C
Huibregtse
Jon M
JM
Gygi
Steven
S
Krug
Robert M
RM
D000998:Antiviral Agents / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism*; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D019204:GTP-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007113:Immunity, Innate / Q000502:physiology*; D009981:Influenza B virus / Q000378:metabolism; D007370:Interferon Type I / Q000378:metabolism*; D013058:Mass Spectrometry; D064547:Myxovirus Resistance Proteins; D020365:RNA Helicases / Q000378:metabolism; D011971:Receptors, Immunologic; D044764:Ubiquitin-Activating Enzymes / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D014452:Ubiquitins / Q000378:metabolism; D019892:eIF-2 Kinase / Q000378:metabolism
D000998:Antiviral Agents; D016207:Cytokines; D007370:Interferon Type I; C579071:MX1 protein, human; D064547:Myxovirus Resistance Proteins; D011971:Receptors, Immunologic; C471538:UBA1 protein, human; D014452:Ubiquitins; C074034:ISG15 protein, human; C471622:UBE2L6 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D019892:eIF-2 Kinase; C452820:RIGI protein, human; D019204:GTP-Binding Proteins; D000071457:DEAD Box Protein 58; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; D044764:Ubiquitin-Activating Enzymes