The UbcH8 ubiquitin E2 enzyme is also the E2 enzyme for ISG15, an IFN-α/β-induced ubiquitin-like protein

Abstract

Ubiquitin-(Ub) like proteins (Ubls) are conjugated to their targets by an enzymatic cascade involving an E1 activating enzyme, an E2 conjugating enzyme, and in some cases an E3 ligase. ISG15 is a Ubl that is conjugated to cellular proteins after IFN-␣͞␤ stimulation. Although the E1 enzyme for ISG15 (Ube1L͞E1 ISG15 ) has been identified, the identities of the downstream components of the ISG15 conjugation cascade have remained elusive. Here we report the purification of an E2 enzyme for ISG15 and demonstrate that it is UbcH8, an E2 that also functions in Ub conjugation. In vitro assays with purified Ub E2 enzymes and in vivo RNA interference assays indicate that UbcH8 is a major E2 enzyme for ISG15 conjugation. These results indicate that the ISG15 conjugation pathway overlaps or converges with the Ub conjugation pathway at the level of a specific E2 enzyme. Furthermore, these results raise the possibility that the ISG15 conjugation pathway might use UbcH8-competent Ub ligases in vivo. As an initial test of this hypothesis, we have shown that a UbcH8-competent Ub ligase conjugates ISG15 to a specific target in vitro. These results challenge the concept that Ub and Ubl conjugation pathways are strictly parallel and nonoverlapping and have important implications for understanding the regulation and function of ISG15 conjugation in the IFN-␣͞␤ response. IFN-␣͞␤ play an essential role in innate immunity and are induced during many types of viral infections (1). Many genes are transcriptionally induced by IFN-␣͞␤, including ISG15 (IFNstimulated gene, 15 kDa) (2, 3). The ISG15 protein is a 15-kDa ubiquitin (Ub)-like protein (Ubl), consisting of two Ub-related domains, Ϸ30% (N-terminal domain) and 36% (C-terminal domain) identical to Ub. ISG15 becomes conjugated to a diverse set of cellular proteins after IFN-␣͞␤ stimulation (4). Although the biochemical consequences of ISG15 conjugation and the fate of the conjugated proteins are not known, it does not appear that ISG15 targets proteins for proteasomal degradation (5, 6).Conjugation of Ub to target proteins requires the cooperative activities of at least three classes of enzymes (7). The ATPdependent E1 enzyme activates Ub by C-terminal adenylation, followed by formation of a high-energy thioester bond between the terminal carboxylate of Ub and the active-site cysteine of E1. Ub is then transferred to the active-site cysteine of one of a number of related E2 enzymes. E3 enzymes then promote transfer of Ub from the E2 to the substrate, resulting in a stable amide bond between -amino groups of lysine side chains and Ub. E3 enzymes are the primary determinants of substrate specificity and can be divided into two classes based on mechanism. HECT E3s accept Ub from the E2 enzyme, again in the form of a thioester adduct, and transfer Ub from their active-site cysteine to the bound substrate (8). RING E3s consist of several subclasses and are either single or multisubunit enzymes that serve as docking proteins for both protein substrates and activated E2 enzymes, with transfe...

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].…”