Interferon-Inducible Ubiquitin E2, Ubc8, Is a Conjugating Enzyme for Protein ISGylation

Abstract

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 interferonsignal 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.

USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis

“…Coexpression of FAT10 with wild-type USE1 resulted in the formation of a USE1 -FAT10 conjugate in intact cells, but this was not the case when the [C 188 A]USE1 active-site mutant was used. Formation of such a conjugate was specifi c for USE1, as a similar coexpression experiment performed with UbcH8, which is a bispecifi c E2 enzyme for ubiquitin and ISG15 22,23 , formed a conjugate with ubiquitin but not with FAT10 ( Fig. 3c ).…”

Expression, regulation and function of the ISGylation system in prostate cancer

“…Similar to ubiquitin, ISG15 is covalently conjugated to cytoplasmic and nuclear proteins, which function in diverse cellular pathways (Zhao et al, 2005). The coupling of ISG15 to its targets is a mechanism similar to the attachment of ubiquitin, and involves the ISG15-specific E1-like activating enzyme, Ube1L (Yuan and Krug, 2001), and the E2 enzyme, UbcH8, which also functions in ubiquitin conjugation (Kim et al, 2004;Zhao et al, 2004). Recently, two ubiquitin ligases, the estrogen-responsive finger protein (EFP) (Zou and Zhang, 2006) and Herc5 (Dastur et al, 2006), have been described to participate in ISG15 ligation.…”

Identification and Herc5-mediated ISGylation of novel target proteins

“…The expression of ISG15 is induced by interferon stimulation and ISG15 is conjugated to various cellular proteins (ISGylation) in a manner similar to ubiquitination that is catalyzed by the sequential action of E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) [4,5]. Target proteins modified with ISG15 [6][7][8], the E1 (UBE1L) and E2 (UbcH8) enzymes functioning in ISGylation [9][10][11], and a de-ISGylating enzyme (UBP43) [12] have been identified, but biological consequences of ISGylation have been studied in only a few cases [13][14][15]. Recently, Efp and Herc5 have been reported to function as E3 ligases for ISGylation [16,17], but there seems to be a difference in function between Efp and Herc5 because Herc5, but not Efp, influences the ISGylation status of whole cellular proteins [16,17].…”