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. 2006 Jul 11;103(28):10735-40.
doi: 10.1073/pnas.0600397103. Epub 2006 Jun 30.

HERC5 is an IFN-induced HECT-type E3 protein ligase that mediates type I IFN-induced ISGylation of protein targets

Joyce Jing Yi Wong  1 , Yuh Fen Pung, Newman Siu-Kwan Sze, Keh-Chuang Chin
Affiliations

HERC5 is an IFN-induced HECT-type E3 protein ligase that mediates type I IFN-induced ISGylation of protein targets

Joyce Jing Yi Wong et al. Proc Natl Acad Sci U S A. .

Abstract

Type I IFNs induce the expression of IFN-stimulated gene 15 (ISG15) and its conjugation to cellular targets. ISGylation is a multistep process involving IFN-inducible Ube1L, UbcH8, and a yet-to-be identified E3 ligase. Here we report the identification of an IFN-induced HECT-type E3 protein ligase, HERC5/Ceb1, which mediates ISGylation. We also defined a number of proteins modified by ISG15 after IFN triggering or HERC5 overexpression. A reduction in endogenous HERC5 by small interfering RNA inhibition blocks the IFN-induced ISG15 conjugation. Conversely, HERC5 coexpression with Ube1L and UbcH8 induces the ISG15 conjugation in vivo independent of IFN stimulation. A targeted substitution of Cys-994 to Ala in the HECT domain of HERC5 completely abrogates its E3 protein ligase activity. Therefore, this study demonstrates that HERC5/Ceb1 is involved in the conjugation of ISG15 to cellular proteins.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Identification and purification of ISG15-associated and/or -modified proteins. (A) Schematic representation of the ISG15 with two copies of FLAG at the N terminus. Mature ISG15 terminates in a conserved GG (bold), and the arrow indicates the cleavage site. ISG15 protein contains two ubiquitin-like domains and is therefore predicted to be ≈15 kDa. Indeed, ISG15 protein runs as 15 kDa in SDS/PAGE. (B–E) Validation of ISG15 conjugation of destrin, cofilin, enolase, and GAPDH. HeLa cells were transfected with plasmids encoding FLAG–ISG15 and treated with IFN-β for 48 h. Total lysates were immunoprecipitated with anti-FLAG M2-conjugated agarose and immunoblotted with antibodies against destrin, cofilin, enolase, and GAPDH.
Fig. 2.
Fig. 2.
HERC5 is an IFN-inducible protein and is covalently modified with ISG15. (A) HERC5 interacts with ISG15 and forms conjugated species with ISG15. HeLa cells were cotransfected with FLAG–HERC5 and Myc–ISG15, and cells were treated (lanes 3 and 4) or not treated (lanes 1 and 2) with IFN-β. Total lysates were prepared and immunoprecipitated with either control (lanes 1 and 3) or FLAG (lanes 2 and 4) antibody and then separated on SDS/PAGE. The protein complexes were detected with either anti-Myc (Left) or anti-FLAG (Right) antibody. (B) Kinetics of IFN-induced HERC5 mRNA expression in HeLa (Left) and A549 (Right) cells. HeLa and A549 cells were either untreated or treated with IFN-β for 6, 12, 24, and 48 h. Total RNAs were isolated, and real-time PCR was performed with HERC5-specific primers. (C) Induction of ISG15 conjugation in HeLa (Left) and A549 (Right) cells after IFN-β treatment. The unstimulated and IFN-stimulated HeLa and A549 cells were harvested at 6, 12, 24, and 48 h. Total lysates were separated in SDS/PAGE and immunoblotted with anti-ISG15 antibody.
Fig. 3.
Fig. 3.
Depletion of HERC5 inhibits the induction of ISG15 conjugation by IFN-β. (A) HERC5 depletion by siRNAs inhibits ISG15 conjugation induced by IFN-β in A549 cells. The A549 cells were transfected with control siRNA (lane 1) or siRNAs against HERC5 (lanes 2 and 3). Twenty-four hours after transfection, A549 cells were treated with IFN-β for another 48 h. Total lysates were harvested and separated in SDS/PAGE. ISG15 conjugates were detected by anti-ISG15 antibody. (B) Suppression of HERC5 mRNA expression in A549 cells by siRNAs. Efficiency of siRNA–HERC5-I and siRNA–HERC5-II in inhibition of HERC5 RNA expression was determined in A549 cells transfected with siRNA control and siRNAs for HERC5 24 h after IFN-β treatment. Expression of HERC5 mRNA was analyzed by real-time PCR with specific primers for HERC5. (C) HERC5 elimination has no effect on the induction of Ube1L and UbcH8 by type I IFN. Total lysates were prepared from A549 cells transfected with control siRNA (lane 1) or siRNAs against HERC5 (lanes 2 and 3) at 24 h after IFN-β treatment. The levels of protein expression of Ube1L and UbcH8 were detected by antibodies against Ube1L (C Upper) and UbcH8 (C Lower), respectively. (D) Suppression of IFN-β-induced HERC5 mRNA expression in HeLa cells constitutively expressing shRNA–HERC5–1-4. Efficiency of shRNA–HERC5–1-4 in inhibition of endogenous HERC5 RNA expression was determined by using real-time PCR with HERC5-specific primers. (E) Inhibition of IFN-β-induced ISG15 conjugation in HeLa cells stably expressing shRNA against HERC5. HeLa cells were stably transfected with vectors expressing shRNA–HERC5–1-4 and selected with 1 μg/ml puromycin. Cells were treated with IFN-β for 48 h, and ISG15 conjugates were analyzed by immunoblotting with anti-ISG15 antibody.
Fig. 4.
Fig. 4.
HERC5, together with Ube1L and UbcH8, induces the FLAG–ISG15 conjugation in HeLa cells without type I IFN treatment. (A) HeLa cells were transfected with expression vector encoding for FLAG–ISG15 plus vectors encoding Ube1L (lane 2), UbcH8 (lane 3), Ube1L and UbcH8 (lane 4), or Ube1L, UbcH8, and HERC5 (lanes 5). Twenty-four hours after transfection, total lysates were prepared, and FLAG–ISG15 conjugates were detected with anti-FLAG antibody. (B–I) HeLa cells were transfected with expression vector encoding for FLAG–ISG15 (lane 1), FLAG–ISG15 plus vector encoding Ube1L and UbcH8 (lane 2), or FLAG–ISG15 construct together with vectors encoding Ube1L, UbcH8, and HERC5 (lanes 3). Total lysates were harvested, FLAG–ISG15 conjugates were immunoprecipitated with anti-FLAG M2-conjugated agarose, and protein complexes were separated in SDS/PAGE and then immunoblotted with antibodies against destrin (B), cofilin (C), enolase (D), GAPDH (E), Hsp27 (F), Hsc70 (G), peroxiredoxin I (H), and peroxiredoxin VI (I).
Fig. 5.
Fig. 5.
Conserved Cys-994 within the HECT domain of HERC5 is essential for its ligase activity in ISGylation. Cys-994 within the HECT domain of HERC5 was mutated to alanine residue. HeLa cells were transfected with vectors expressing 3Myc–ISG15 (lane 2); 3Myc–ISG15, Ube1L, and UbcH8 (lane 3); 3Myc–ISG15, Ube1L, UbcH8, and wild-type FLAG–HERC5 (lane 4); and 3Myc–ISG15, Ube1L, UbcH8, and FLAG–HERC5–C994A (lane 5). Forty-eight hours after transfection, cell lysates were collected, and Myc–ISG15 conjugates were detected by immunoprecipitation followed by immunoblotting with anti-Myc antibody (Upper). The expression of wild-type and mutant HERC5 was detected with anti-FLAG antibody (Lower).
Fig. 6.
Fig. 6.
HERC5 interacts with ISGylated substrates, Hsc70, and thioredoxin reductase. (A) HeLa cells were transfected with HA-Hsc70 (lane 2), FLAG–HERC5 (lane 3), or FLAG–HERC5 plus HA-Hsc70 (lane 4). Total lysates were made and immunoprecipitated with anti-FLAG agarose. The immunocomplexes were separated and immunoblotted with anti-HA (Top) for detection of Hsc70 or anti-FLAG (Middle) for HERC5. The total amount of Hsc70 was confirmed by immunoblotting of cell lysate with anti-HA antibody (Bottom). (B) Same as above, except that the HA-tagged form of thioredoxin reductase (HA-THR) was used in the transfection.
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References

    1. Decker T., Muller M., Stockinger S. Nat. Rev. Immunol. 2005;5:675–687. - PubMed
    1. Perry A. K., Chen G., Zheng D., Tang H., Cheng G. Cell Res. 2005;15:407–422. - PubMed
    1. Smith P. L., Lombardi G., Foster G. R. Mol. Immunol. 2005;42:869–877. - PubMed
    1. Levy D. E., Darnell J. E., Jr. Nat. Rev. Mol. Cell Biol. 2002;3:651–662. - PubMed
    1. Stark G. R., Kerr I. M., Williams B. R., Silverman R. H., Schreiber R. D. Annu. Rev. Biochem. 1998;67:227–264. - PubMed

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