Herc5, an Interferon-induced HECT E3 Enzyme, Is Required for Conjugation of ISG15 in Human Cells

Abstract

ISG15 is an interferon (IFN)-␣/␤-induced ubiquitin-like protein that is conjugated to cellular proteins during innate immune responses to viral and bacterial infections.A recent proteomics study identified 158 human proteins targeted for ISG15 conjugation, including the ISG15 E1 and E2 enzymes (Ube1L and UbcH8, respectively) and a HECT E3 enzyme, Herc5. Like the genes encoding Ube1L and UbcH8, expression of Herc5 was also induced by IFN-␤, suggesting that Herc5 might be a component of the ISG15 conjugation system. Consistent with this, small interfering RNAs targeting Herc5 had a dramatic effect on overall ISG15 conjugation in human cells, abrogating conjugation to the vast majority of ISG15 target proteins in vivo. In addition, co-transfection of plasmids expressing ISG15, Ube1L, UbcH8, and Herc5 resulted in robust ISG15 conjugation in non-IFN-treated cells, while the active-site cysteine mutant of Herc5 or a mutant lacking the RCC1 repeat region did not support ISG15 conjugation. These results demonstrate that Herc5 is required for conjugation of ISG15 to a broad spectrum of target proteins in human cells.Type 1 interferons play an essential role in innate immunity (1). One of the many genes strongly activated by IFN 4 -␣/␤ encodes ISG15, a 15-kDa ubiquitin-like protein (Ubl) (2, 3). Like ubiquitin (Ub), Ubls are linked to target proteins via isopeptide bonds between their terminal carboxyl group and lysine side chains of target proteins (4). The fact that ISG15 is expressed and conjugated in IFN-␣/␤-stimulated cells and lipopolysaccharide-stimulated cells implies that ISG15 conjugation is likely to mediate an important component of the innate immune response. This is supported by the finding that the influenza B virus NS1B protein specifically blocks ISG15 conjugation (5).The biochemical effect of ISG15 on target proteins is unknown; however, the recent identification of a large number of target proteins (6) provides opportunities for determining both the function of ISG15 and its role in the innate immune response. Also essential for functional studies is the identification of the complete set of enzymes required for ISG15 conjugation. As with Ub conjugation, it is presumed that a cooperating set of E1, E2, and E3 enzymes, in addition to possible accessory factors, will be required for ISG15 conjugation. The ISG15 E1 and E2 enzymes have been identified. Ube1L is a single-subunit enzyme 62% similar to the Ub E1 enzyme (5), and UbcH8 is the major, if not exclusive, E2 enzyme for ISG15 (7,8). The genes encoding both Ube1L and UbcH8 are, like ISG15, transcriptionally activated by IFN-␣/␤ (5, 7-9), suggesting that the entire conjugation system might be coordinately regulated. A candidate E3 enzyme for ISG15 conjugation emerged from mass spectrometry-based identification of ISG15 target proteins (6). Proteomics analyses of SUMO-and Ub-conjugated proteins have shown that enzymatic components of Ub/Ubl conjugation pathways are often auto-conjugated (10, 11), and consistent with this, Ube1L and UbcH8 were identified ...

Identification and Herc5-mediated ISGylation of novel target proteins

“…These results agree well with results of a previous study showing that Herc5 is an accelerator of a broad range of ISG15 conjugation [17]. The construction of an in vitro ISGylation system containing recombinant Herc5 together with ISG15, UBE1L and UbcH8 will verify the speculation that Herc5 functions as a general E3…”

“…Efp and Herc5 have been reported to be E3 ligases for ISGylation [16,17], although they also function as ubiquitin E3 ligases [18,20]. We carried out experiments to determine which 7 E3 ligase stimulates ISGylation of four target proteins newly identified in this study, XPD (ERCC2), STK38, RGS3 isoform 1 and α-tubulin.…”

“…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].…”

The protein kinase IKKε can inhibit HCV expression independently of IFN and its own expression is downregulated in HCV-infected livers

“…59 ISG15 forms covalent interaction with proteins but instead of targeting them for proteasomal degradation, like ubiquitin, it provokes posttranslational modifications that play a role on half-life, cellular localization, and protein activity. ISG15 conjugaison of proteins (or ISGylation) requires the activity of the E1-(Ube1L), E2-(UbcH8), and E3-CEB1/HERC5 conjugating enzymes, [59][60][61] whereas the deconjugation process is performed by the specialized ubiquitin protease USP18. The importance of USP18 in innate immunity to viral infection was demonstrated using USP18-deficient mice, which were resistant to lymphocytic choriomeningitis virus or vesicular stomatitis virus .…”

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

“…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. A major feature of the ISGylation system is the upregulation of all constituents by type I IFNs.…”