Interferon-inducible ubiquitin E2, Ubc8, is a conjugating enzyme for protein ISGylation.
Protein ISGylation is unique among ubiquitin-like conjugation systems in that the expression and conjugation processes are induced by specific stimuli, mainly via the alpha/beta interferon signaling pathway. It has been suggested that protein ISGylation plays a special role in the immune response, because of its interferon-signal dependency and its appearance only in higher eukaryotic organisms. Here, we report the identification of an ISG15-conjugating enzyme, Ubc8. Like other components of the protein ISGylation system (ISG15, UBE1L, and UBP43), Ubc8 is an interferon-inducible protein. Ubc8 clearly mediates protein ISGylation in transfection assays. The reduction of Ubc8 expression by small interfering RNA causes a decrease in protein ISGylation in HeLa cells upon interferon treatment. Neither UbcH7/UbcM4, the closest homologue of Ubc8 among known ubiquitin E2s, nor the small ubiquitin-like modifier E2 Ubc9 supports protein ISGylation. These findings strongly suggest that Ubc8 is a major ISG15-conjugating enzyme responsible for protein ISGylation upon interferon stimulation. Furthermore, we established an assay system to detect ISGylated target proteins by cotransfection of ISG15, UBE1L, and Ubc8 together with a target protein to be analyzed. This method provides an easy and effective way to identify new targets for the ISGylation system and will facilitate related studies.
The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
Keun Il
KI
Kim
Nadia V
NV
Giannakopoulos
Herbert W
HW
Virgin
Dong-Er
DE
Zhang
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D016207:Cytokines / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D006801:Humans; D007372:Interferons / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D034622:RNA Interference; D050794:STAT1 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D015534:Trans-Activators / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D044764:Ubiquitin-Activating Enzymes / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014452:Ubiquitins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016207:Cytokines; D004268:DNA-Binding Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; C494080:Stat1 protein, mouse; D015534:Trans-Activators; D014452:Ubiquitins; C074034:ISG15 protein, human; D007372:Interferons; D044763:Ubiquitin-Conjugating Enzymes; C409686:ubiquitin-conjugating enzyme UBC8; D044764:Ubiquitin-Activating Enzymes