Identification and Herc5-mediated ISGylation of novel target proteins

Abstract

ISG15, a protein containing two ubiquitin-like domains, is an interferon-stimulated gene product that functions in antiviral response and is conjugated to various cellular proteins (ISGylation) upon interferon stimulation. ISGylation occurs via a pathway similar to the pathway for ubiquitination that requires the sequential action of E1/E2/E3: The E1 (UBE1L), E2 (UbcH8), and E3 (Efp/Herc5) enzymes for ISGylation have been hitherto identified. In this study, we identified six novel candidate target proteins for ISGylation by a proteomic approach. Four candidate target proteins were demonstrated to be ISGylated in UBE1L-and UbcH8-dependent manners, and ISGylation of the respective target proteins was stimulated by Herc5. In addition, Herc5 was capable of binding with the respective target proteins. Thus, these results suggest that Herc5 functions as a general E3 ligase for protein ISGylation.

Negative regulation of ISG15 E3 ligase EFP through its autoISGylation

“…However, EFP has little effect on the total ISGylation when co-transfected with ISG15, UBE1L, and UbcH8 into 293T cells [11]. The direct comparison between EFP and Herc5 are reported by a recent paper [19]. Among the tested four targets, EFP can increase the ISGylation of tubulin as well as Herc5 and weakly increase the conjugation of two other targets.…”

“…Among the tested four targets, EFP can increase the ISGylation of tubulin as well as Herc5 and weakly increase the conjugation of two other targets. However, Herc5 can increase the ISGylation of all four targets [19]. We also tested the effect of Herc5 on the ISGylation of 14-3-3σ.…”

Activation of Double-stranded RNA-activated Protein Kinase (PKR) by Interferon-stimulated Gene 15 (ISG15) Modification Down-regulates Protein Translation

“…We therefore investigated whether PKR is modified by ISG15. UBE1L, mUbcH8, His 6 -tagged ISG15, and HA-tagged PKR were expressed in HEK293T cells together with the nonspecific ISG15 ligase Herc5 to improve ISGylation (11)(12)(13). ISGylated proteins were purified by Ni-NTA-agarose chromatography and subjected to Western blotting against an anti-HA antibody to detect the ISGylation of HA-PKR (Fig.…”

“…The ubiquitin E2 enzymes UbcH6 and UbcH8 also function as ISG15-conjugating enzymes (6 -8). In contrast to ubiquitin ligases, only a few ISG15 ligases have been identified, including EFP (9), HHARI (10), Herc5 (11)(12)(13), and Herc6 (14,15). USP18 (UBP43) is an isopeptidase that removes ISG15 from its substrates (16).…”

Herc5 Attenuates Influenza A Virus by Catalyzing ISGylation of Viral NS1 Protein

“…Unlike ubiquitination, ISGylation typically does not promote degradation of the target proteins. A couple of proteomics studies have identified .100 cellular proteins as potential targets of ISGylation (32)(33)(34)(35). These proteins cover a wide spectrum of biological processes, including transcriptional regulation, signal transduction, inflammation, and control of cell growth.…”