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Herc5, an interferon-induced HECT E3 enzyme, is required for conjugation of ISG15 in human cells

Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, United States
Journal of Biological Chemistry (Impact Factor: 4.6). 03/2006; 281(7):4334-8. DOI: 10.1074/jbc.M512830200
Source: PubMed

ABSTRACT ISG15 is an interferon (IFN)-alpha/beta-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-beta, 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.

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    • "For example, ISG15 (first reported in 1978) contains two ubiquitin-like domains linked by a hinge, and the C-terminal LRLRGG motif is responsible for its conjugating onto target proteins. ISG15 and its E1 activating enzyme Ube1L, E2 conjugating enzyme UbcH8, E3 ligase Herc5 are all robustly induced upon viral infection or lipopolysaccharide (LPS) treatment, causing the ISGylation of a wide spectrum of proteins [18] [19] [20] [21] [22]. The functional consequences of ISGylation are currently under intensive investigation . "
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    • "There was no significant difference in the viability of cells expressing HERC5 + CS compared to the control cells (92.0% ± 4.3% SD versus 90.2% ± 4.4% SD respectively; P > 0.05, paired t test). Western blot analysis of the cell lysates revealed the presence of ISG15 conjugates in cells transfected with the CS and HERC5, but not the cells transfected with the CS and empty vector or HERC5-C994A, thus supporting previous data (Figure 4B, upper blot)(Dastur et al., 2006; Wong et al., 2006). We also verify that HERC5, but not HERC5-C994A, can conjugate ISG15 to itself (Figure 4B, lower blot). "
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    • "DNA encoding the isolated HECT domain of mHERC6 (aminoacids (aa) 658–1003) and the RCC1-like domain truncation mutant (aminoacids (aa) 371–1003) were amplified via PCR and cloned into the pTriEx-2 vector. Plasmids encoding human Ube1l and human UbcH8 were described previously [21]. Murine and human ISG15 were expressed using pcDNA3.1 (Invitrogen). "
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