Abstract
The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3sigma protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3sigma in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3sigma in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations.
Highlights
ISG15 is composed of two domains, each of which bears sequence identity to ubiquitin (33 and 32% for the N- and C-terminal domains, respectively) [7]
We hypothesized that ISG15 E3 ligases are UBC8-interacting ubiquitin E3 ligases that may be encoded by IFN-inducible genes
Research on ISGylation has been accelerated by the identification of the enzymes involved in the ISG15 conjugation process, including the ISG15 E1 enzyme UBE1L, the E2 enzyme UBC8, and the de-ISGylating enzyme UBP43 [12, 14, 16, 17]
Summary
ISG15 is composed of two domains, each of which bears sequence identity to ubiquitin (33 and 32% for the N- and C-terminal domains, respectively) [7]. Using the small interfering RNA (siRNA) approach, both groups indicated that UBC8 is a predominant ISG15 E2 enzyme for IFN-induced protein ISGylation in HeLa cells. EFP Is Up-regulated upon IFN Treatment—Because the primary and tertiary structures of ISG15 are similar to those of ubiquitin [30], the mechanism of ISGylation is expected to be similar to that of protein ubiquitylation and is predicted to involve the activity of the E1, E2, and E3 enzymes.
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