Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Proc Natl Acad Sci U S A. 2006 Jul 11; 103(28): 10735–10740.
Published online 2006 Jun 30. doi: 10.1073/pnas.0600397103
PMCID: PMC1484417
PMID: 16815975

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

Joyce Jing Yi Wong,* Yuh Fen Pung,* Newman Siu-Kwan Sze,† and Keh-Chuang Chin*‡§
Author information Article notes Copyright and License information PMC Disclaimer

Associated Data

Supplementary Materials

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.

Keywords: ISG15, Ceb1, innate immunity, antiviral proteins

Type I IFNs (IFN-α/β) play an essential role in both innate antiviral and adaptive immune responses and are rapidly produced in response to microbial infection (1–3). They exert signals through the activation of the Janus kinase–signal transducer and activator of transcription pathway that mediates rapid induction of IFN-stimulated genes (ISGs) (4, 5). ISG15 is one of the most strongly induced genes after IFN treatment (6, 7) and is also significantly induced by viral infection (8, 9) and LPS treatment (10, 11). The ISG15 protein starts with two ubiquitin-related domains that have ≈27% sequence identity to ubiquitin and terminate in a conserved 152LRLRGG157 ubiquitin C-terminal motif. This study suggests that ISG15 could act in a similar way to ubiquitin and other ubiquitin-like proteins such as SUMO by forming an isopeptide bond with cellular proteins (6, 12, 13). The crystal structure of ISG15 revealed that ISG15 consists of two domains with ubiquitin-like folds joined by a linker sequence (14).

Conjugation of ISG15 to cellular proteins occurs in a parallel but distinct mechanism to that of ubiquitin (15–17). The E1 enzyme for ISG15, Ube1L, is a single-subunit enzyme and is identified in vitro by its ability to catalyze the formation of a thioester bond between ISG15 and Ube1L (17, 18). The Ube1L protein is highly similar to the E1 enzyme for ubiquitin at the protein level. However, this protein does not form a conjugate with ubiquitin, indicating that Ube1L is an E1 enzyme for the ISG15 conjugation system (ISGylation). Influenza B virus blocks protein ISGylation by inhibiting the activation step through the interaction of the NS1B viral protein with ISG15 (18). This finding was the first suggestion that ISGylation might be important for protecting cells from viral infection.

Two groups recently found that a member of the ubiquitin E2-conjugating enzyme family, UbcH8, is involved in the ISGylation (19, 20). Like ISG15 and Ube1L, the expression of UbcH8 is also induced by IFN (21). The suppression of UbcH8 protein expression by RNA interference is shown to dramatically inhibit the total level of IFN-induced conjugation of ISG15 to cellular proteins (19, 20).

In the ubiquitin and ubiquitin-like proteins (ublps) system, E3 enzymes play a critical role in transferring ubiquitin or ublps such as SUMO from the E2-conjugating enzyme to a specific substrate. The substrate protein interacts directly with a specific domain in the E3 enzymes or through an adaptor associated with the E3 enzyme (22). E3 enzymes are known to be divided into two groups: HECT (homologous to E6-AP C terminus) and RING (really interesting new genes) proteins (22–25). Whereas RING-type proteins transfer ubiquitin directly from an E2 to a target, the HECT-type protein forms a thioester bond with ubiquitin or ublps by its active cysteine residue before transferring it to a substrate (25). The identity of the E3 enzyme(s) in the ISG15 conjugation system has not been resolved.

Here we report the identification of target proteins conjugated with ISG15 and present data supporting the contention that the covalent modification of cellular proteins by ISG15 is regulated by an IFN-inducible HECT domain-containing E3 protein ligase, HERC5, which itself is also a target for modification by ISG15. HERC5 catalyzes ISGylation by means of a catalytic cysteine residue at position 994 in the HECT domain. Moreover, HERC5 is found to be sufficient for inducing ISGylation in the absence of IFN. Knock-down of HERC5 by small interfering RNA (siRNA) results in the inhibition of IFN-induced ISG15 conjugation. Thus, HERC5 is an IFN-induced E3 protein ligase that mediates ISGylation.

Results

Identification and Purification of FLAG–ISG15-Associated and/or -Modified Proteins.

To search for potential E3 enzyme(s) as well as proteins that were physically associated or covalently conjugated with ISG15, we generated A549 cell lines stably expressing ISG15 with two copies of FLAG tag (Fig. 1A). The FLAG tags were inserted at the N terminus of ISG15 because of the involvement of the conserved C terminus diglycine motif in conjugation with cellular proteins (Fig. 1A). The FLAG–ISG15 retains its conjugation activity to cellular proteins after 48 h of IFN-β treatment as compared with untreated control (Fig. 7A, which is published as supporting information on the PNAS web site). These data indicate that FLAG tags inserted at the N terminus of ISG15 do not disrupt the conjugation to cellular proteins after IFN-β treatment.

An external file that holds a picture, illustration, etc. Object name is zpq0280627630001.jpg

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.

We next carried out large-scale isolation of ISG15-associated/conjugated proteins by anti-FLAG immunoaffinity purification (Fig. 7B). Control A549 fractions were also analyzed to identify nonspecific, copurified proteins and hence eliminate false positives. A total of 174 unique candidate proteins, not found in control fractions, and represented by at least two peptides in the tandem MS analysis, were identified. The ISG15 target proteins that fulfilled these stringent criteria are listed in Table 1, which is published as supporting information on the PNAS web site.

Twenty-six target proteins were selected for further validation of their conjugation or association with ISG15. Twenty-four of 26 putative ISG15 target proteins were found to form conjugates with ISG15 after IFN-β treatment (Table 1, bold). Two other ISG15-copurified proteins, Hsp70 and pICln, were not directly conjugated to ISG15 but formed a specific, noncovalent interaction (data not shown). Of the proteins that were substrates for ISG15 conjugation, a number, such as destrin and cofilin, were found to conjugate with a single ISG15 as they migrated at the expected molecular masses for destrin*FLAG–ISG15 (Mr ≈ 37 kDa) (Fig. 1B, lane 2) and cofilin*FLAG–ISG15 (Mr ≈ 37 kDa) (Fig. 1C, lane 2). On the other hand, some proteins, such as enolase and GAPDH, existed as multiply conjugated species that were possibly modified by one or more ISG15s (Fig. 1 D and E, lane 2). At least two isoforms of enolase-conjugated species were observed, migrating at the positions expected for one and three ISG15s (Fig. 1D, lane 2). Similar to enolase, GAPDH also displayed a number of conjugated species that migrated at the positions expect for one and two ISG15s in SDS/PAGE (Fig. 1E, lane 2). In addition to conjugation, we also observed noncovalent association between ISG15 and cellular substrates (Fig. 1 B–E, shown by arrowhead). Thus, we have identified a large number of cellular proteins that form either single or multiple conjugation and noncovalent interactions with ISG15.

HERC5/Ceb1 Is Copurified with ISG15, and Its Expression Is Regulated by Type I IFN.

Conjugation of ISG15 with cellular proteins is catalyzed by the E1 activating enzyme Ube1L and the E2-conjugating enzyme UbcH8 (17). Thus far, no E3 enzyme(s) has been identified. In ubiquitin system, E3 enzymes, with either a HECT domain or a RING domain, play a critical role in recruiting the ubiquitin-loaded E2, recognizing specific substrates and facilitating the transfer of the ubiquitin from the E2 to the lysine residue in the substrates (22). Among all ISG15-associated/conjugated candidate proteins, we noticed that a protein known as HERC5 (or Ceb1) contains a HECT-type domain at its C terminus (Fig. 8, which is published as supporting information on the PNAS web site) (26). As shown in Table 2, which is published as supporting information on the PNAS web site, HERC5 was represented by five individual peptides from tandem MS sequencing and was not found in the control sample, indicating that this protein is specifically copurified with FLAG–ISG15.

The primary sequence of HERC5 revealed the presence of RCC1-like (regulator of chromosome condensation-1) and HECT domains that span residues 209–258 and 676-1024, respectively (Fig. 8A) (26). The HECT domain was previously demonstrated to interact with a ubiquitin-conjugating enzyme (23). Within the HECT domain of HERC5, a conserved cysteine residue residing in all known HECT-type protein ligases was also found and located at position 994 (Fig. 8B) (26). Because HERC5/Ceb1 possesses all of the unique structural features of a mammalian E3 protein ligase, we hypothesized that HERC5 functions as an E3 protein ligase in ISGylation.

Because HERC5 is identified by the pulling down of ISGylated proteins and ISG15-associated proteins, we first determined whether ISG15 binds HERC5 or forms covalent conjugated species with HERC5. HeLa cells were cotransfected with FLAG–HERC5 and Myc–ISG15 (Fig. 2A, lanes 1–4), and cells were treated with (Fig. 2A, lanes 3 and 4) or without (Fig. 2A, lanes 1 and 2) IFN-β. We performed either anti-FLAG (Fig. 2A, lanes 2 and 4) or control (Fig. 2A, lanes 1 and 3) immunoprecipitation followed by anti-Myc or anti-FLAG Western blotting to determine whether higher-molecular-weight forms of HERC5 or free forms of ISG15 could be detected. As shown in Fig. 2A, we saw multiple species of high molecular forms of HERC5 in IFN-β-treated cells expressing both FLAG–HERC5 and Myc–ISG15 by anti-Myc and anti-FLAG Western blotting, respectively. Moreover, we saw free forms of ISG15 copurified with HERC5 in both untreated and IFN-β-treated cells (Fig. 2A Left). These data indicate that HERC5 physically associates with ISG15 and itself is a target for modification by ISG15.

An external file that holds a picture, illustration, etc. Object name is zpq0280627630002.jpg

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.

We then sought to examine whether type I IFN induces HERC5 mRNA expression in both HeLa and A549 cell lines because the expression of Ube1L and UbcH8 has previously been shown to be induced by IFN-β (21). Both HeLa and A549 cells were treated with IFN for 6, 12, 24, and 48 h. As shown in Fig. 2B, we detected basal levels of HERC5 mRNA in both untreated HeLa (Left) and A549 (Right) cells. Upon IFN-β stimulation, HERC5 mRNA was rapidly induced in both cell lines after 6 h (Fig. 2B). At 12 h there were ≈30-fold increments in HERC5 mRNA expression levels compared with their respectively untreated controls for both HeLa and A549 cells (Fig. 2B). The mRNA for HERC5 was continuously expressed at high levels at 24 and 48 h after IFN-β treatment as compared with the controls (Fig. 2B). We also measured the induction of ISG15 conjugates in both HeLa (Fig. 2C Left) and A549 (Fig. 2C Right) cells upon IFN-β treatment. In contrast to the kinetics of HERC5 mRNA expression, ISG15 protein conjugates were only detected in both HeLa and A549 cells at 24 h after IFN-β treatment (Fig. 2C, lane 4) and were continually observed at 48 h (Fig. 2C, lane 5). Together, these data indicated that HERC5 is an IFN-inducible protein. Also, the strong induction of HERC5 mRNA expression by IFN-β before ISGylation is paralleled to the observations made for Ube1L and UbcH8. Thus, we hypothesized that HERC5 could function as a protein ligase in concert with Ube1L and UbcH8 to promote ISGylation upon IFN-β treatment.

Depletion of Endogenous HERC5 Inhibits ISGylation upon Type I IFN Treatment.

To investigate the role of HERC5 in ISGylation, we used siRNA technology to suppress the expression of endogenous HERC5 mRNA in A549 cells. Two small siRNAs, namely siRNA–HERC5-I and siRNA–HERC5-II, which target nucleotide sequences 536–554 bp and 715–735 bp, respectively, in HERC5 were used. A549 cells were transfected with either control siRNA or siRNAs against HERC5. After 48 h of IFN-β treatment, total lysates were made and separated in SDS/PAGE. ISG15 conjugates were detected by immunoblotting with an antibody against endogenous ISG15. A dramatic reduction in the expression of ISG15 conjugates was observed in A549 cells transfected with the two siRNAs against HERC5 compared with control siRNA (Fig. 3A). As shown in Fig. 3B, both the siRNAs against HERC5 were effective in reducing HERC5 mRNA expression by 80% after 24 h of IFN-β treatment (Fig. 3B), but not its closest homologue, HERC6 (Fig. 9A, which is published as supporting information on the PNAS web site). It is crucial to note that the reduction in HERC5 mRNA expression had no effect on the expression of ISG15 (Fig. 3A), Ube1L, or UbcH8 (Fig. 3C) protein levels. Thus, these results indicate that HERC5 is a major E3 protein ligase whose induction by IFN-β is necessary for catalyzing ISGylation in A549 cells.

An external file that holds a picture, illustration, etc. Object name is zpq0280627630003.jpg

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.

Next we sought to test whether the inducibility of endogenous ISG15 conjugates by IFN-β in HeLa cells is affected by a short hairpin RNA (shRNA)-mediated reduction in HERC5. A shRNA expression construct, designated as shRNA–HERC5–1-4, was generated. This shRNA was chosen to target a separate region in HERC5, 1,606–1,639 bp. HeLa cells were transfected with shRNA–HERC5–1-4, and cells stably expressing shRNA against HERC5 were then selected by puromycin treatment. The expression of HERC5 mRNA after IFN-β treatment was examined by real-time PCR using primers specific for HERC5. HERC5 mRNA was found to be significantly reduced in HeLa cells constitutively expressing shRNA against HERC5 compared with control cells after IFN-β treatment (Fig. 3D), but not its closest homologue, HERC6 (Fig. 9B). The reduction of HERC5 expression in HeLa cells also significantly impaired the induction of endogenous ISG15 conjugates after 48 h of IFN-β treatment (Fig. 3E). Taken together, these results indicate that HERC5 plays an essential role in catalyzing IFN-β-induced ISGylation in both HeLa and A549 cells.

Coexpression of HERC5 with Ube1L and UbcH8 Restores ISG15 Conjugates in Vivo Without Type I IFN for Induction.

Because HERC5 expression is induced by IFN, we tested whether overexpression of HERC5 in combination with Ube1L and UbcH8 would lead to ISGylation in the absence of IFN-β. HeLa cells transfected with either Ube1L (Fig. 4A, lane 2) or UbcH8 (Fig. 4A, lane 3) expressed no ISG15 conjugates. A partial expression of ISG15 conjugates was observed in HeLa cells transfected with Ube1L and UbcH8 (Fig. 4A, lane 4), which may have been because of a low level of HERC5 expression in HeLa cells (refer to RNA work in Fig. 2B Left). When HERC5 is overexpressed, the production of ISG15 conjugates by HeLa cells is dramatically enhanced (Fig. 4A, lanes 5). These data strongly implicated HERC5 as an E3 protein ligase whose overexpression is sufficient to drive ISGylation.

An external file that holds a picture, illustration, etc. Object name is zpq0280627630004.jpg

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).

Next we examined a number of proteins known to be ISGylated after IFN-β treatment, for conjugation with ISG15 in HeLa cells overexpressing HERC5. HeLa cells were transfected with FLAG–ISG15 alone (Fig. 4 B–I, lane 1), FLAG–ISG15 plus vectors expressing Ube1L and UbcH8 (Fig. 4 B–I, lane 2), or HERC5, Ube1L, and UbcH8 (Fig. 4 B–I, lane 3). Cells were harvested after 48 h. FLAG–ISG15 conjugates were immunoprecipitated with anti-FLAG antibody. The protein complexes were separated in SDS/PAGE and detected with specific antibodies. All of the target proteins examined were found to be covalently conjugated with ISG15 when HERC5 is overexpressed (Fig. 4 B–I, lanes 3). Three target proteins, destrin, enolase, and GAPDH, were observed to have low levels of conjugation in HeLa cells transfected with Ube1L and UbcH8 alone (Fig. 4 B, D, and E, lane 2). However, their levels of conjugation were dramatically enhanced in cells transfected with HERC5, Ube1L, and UbcH8 (Fig. 4 B, D, and E, lane 3). Interestingly, the presence of multiple conjugated species for both enolase and GAPDH were also observed after HERC5 overexpression, indicating that HERC5 is essential for both single and multiple ISGylation in target proteins (Fig. 4 D and E, lane 3). In contrast to enolase and GAPDH, the other five target proteins tested (cofilin, Hsp27, Hsc70, peroxiredoxin I, and peroxiredoxin VI) displayed no detectable ISGylation in HeLa cells transfected with only Ube1L and UbcH8 (Fig. 4 C and F–I, lane 2). These proteins all exhibited significant levels of ISG15 conjugation in the HeLa cells overexpressed with HERC5 (Fig. 4 C and F–I, lane 3). Together, these data indicate that HERC5 is an E3 protein ligase involved in ISGylation. Both single and multiple conjugations in target proteins require the expression of HERC5. Once expressed, the enzymatic activity of HERC5 is not dependent on type I IFNs.

Cys-994 Within the HECT Domain of HERC5 Is Essential for Its Protein Ligase Activity in ISGylation.

All HECT-type ubiquitin protein ligases contain a conserved active cysteine residue that forms an intermediate bond with ubiquitin before transferring it to the substrates (Fig. 8B). To further demonstrate that HERC5 is an E3 protein ligase for ISGylation, the putative active cysteine residue (C994), based on conservation with other HECT domain-containing E3s, was mutated to an alanine residue by site-specific mutagenesis. HeLa cells were cotransfected with Myc–ISG15 and either wild-type FLAG–HERC5 (Fig. 5, lane 4) or mutant FLAG–HERC5 (Fig. 5, lane 5). This point mutation in the cysteine residue (C994) completely abolishes its E3 activity in ISGylation (Fig. 5 Upper, compare lanes 4 and 5). The expression levels of wild-type and mutant HERC5 proteins were found to be comparable (Fig. 5 Lower). These data demonstrate that HERC5 acts as an E3 protein ligase in ISGylation by means of a conserved cysteine residue, C994, found in the HECT domain.

An external file that holds a picture, illustration, etc. Object name is zpq0280627630005.jpg

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).

HERC5 Interacts with ISGylated Substrates, HSC70, and Thioredoxin Reductase.

To further support that HERC5 is a protein ligase for ISG15, we next tested whether HERC5 interacts with ISGylated substrates, Hsc70, and thioredoxin reductase. HeLa cells were transfected with FLAG–HERC5 (Fig. 6, lane 3), ISGylated substrate (Fig. 6, lane 2), FLAG–HERC5 plus HA-Hsc70 (Fig. 6A, lane 4) or HA–thioredoxin reductase (Fig. 6B, lane 4). Cell extracts were immunoprecipitated with an anti-FLAG monoclonal antibody. The immunoprecipitates were then subjected to SDS/PAGE and immunoblotted with antibodies against FLAG or hemagglutinin (HA). As shown in Fig. 6, the antibody against FLAG precipitated FLAG–HERC5 in cells expressing FLAG–HERC5 (Fig. 6 Middle, lanes 3 and 4), but not in the control cells (Fig. 6 Middle, lanes 1 and 2). The FLAG antibody also precipitated Hsc70 (Fig. 6A Top, lane 4) or thioredoxin reductase (Fig. 6B Top, lane 4). The expression of cellular substrates was the same by immunoblotting cell lysates with an HA antibody (Fig. 6 Bottom, lanes 2 and 4). These results suggest that HERC5 acts as an E3 protein ligase for ISGylation by recruiting cellular substrates for modification by ISG15, which is also found to be associated with HERC5 (Fig. 2A).

An external file that holds a picture, illustration, etc. Object name is zpq0280627630006.jpg

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.

Discussion

HERC5 Is an IFN-Inducible Protein Ligase for ISG15 Conjugation System.

We have described the properties of HERC5 identified through its interaction with ISG15. This interaction was detected by using an affinity-based purification scheme involving an antibody against FLAG–ISG15 in A549 cells treated with IFN-β. HERC5 is a member of a group of related proteins known as the HERC family (27, 28). The human HERC family currently consists of six members, HERC1 to HERC6, and is characterized by the presence of a HECT domain and one or more RCC1-like domains (RLDs). Proteins with a HECT domain are now thought to act as E3 protein ligase for ubiquitin and ublps, such as E6-AP and Nedd4 (29). Here we have provided evidence suggesting that HERC5 is an IFN-inducible E3 protein ligase that is required and sufficient for the ISG15 conjugation system in vivo. First, a knock-down of HERC5 expression by RNA interference technology completely inhibited the induction of ISG15 conjugation induced by IFN. Second, HERC5 coexpression with the Ube1L and UbcH8 suffices to mediate ISG15 conjugation in vivo even if in the absence of IFN treatment. Third, a mutation of a conserved cysteine residue in the HECT domain of HERC5 to an alanine residue abolished its protein ligase activity in vivo. This result is consistent with the hypothesis that C994 is important for transferring ISG15 from UbcH8 to specific target proteins. Fourth, HERC5 interacts with ISG15 and ISGylated substrates such as Hsc70 and thioredoxin reductase. The ability of HERC5 to interact with both ISG15 and cellular substrate is consistent with the hypothesis that HERC5 could act as an E3 protein ligase for ISGylation. In addition, we find that knock-down of Ube1, E1 enzyme for the ubiquitination system, has no affect on the ISGylation, and this result rules out the possibility that HERC5 acts an E3 enzyme for ubiquitination by activating an uncharacterized E3 enzyme for ISGylation and inducing ISGylation (Fig. 10, which is published as supporting information on the PNAS web site). The finding that HERC5 is involved in ISGylation in this study was also demonstrated by an independent group while this article was under review (30). One of the challenging tasks is to express HERC5 for promotion of ISGylation in vitro; however, we and Dastur et al. (30) had some difficulty expressing either the full-length or the HECT domain of HERC5 in the bacterial system, and this issue remains to be resolved.

The N-terminal region of HERC5 possesses at least one RLD. RLDs have been shown to interact with several proteins, and it is possible that the RLDs serve as the regions for recruiting specific protein substrates. In addition, the middle region, between RLDs and the HECT domain of HERC5, displays no obvious homology to any known proteins. This region may also serve a role in recognizing target proteins. Further structure–function studies in HERC5 will be needed to provide insight into the specificity of HERC5 in recognizing its target proteins.

Target Proteins Modified by HERC5.

By using a combination of affinity purification and MS, we identified 174 candidate proteins that were covalently conjugated or interacted with ISG15 upon IFN treatment. Of 27 target proteins examined, 24 were identified to conjugate with ISG15, and the other three proteins were found to interact with ISG15 (Table 1 and data not shown). Given that most of the candidates we chose to pursue were modified by the ISG15 conjugation pathway, we are highly confident that our large data set represents bona fide substrates for ISG15 in vivo.

Eight putative IFN-inducible ISG15 targets (MxA, IFIT1, signal transducer and activator of transcription 1, Ube1L, tryptophanyl tRNA synthetase, TRIM21, gelsolin, and HERC5) are identified (4, 5). We chose four of the IFN-induced proteins (MxA, IFIT1, signal transducer and activator of transcription 1, and tryptophanyl tRNA synthetase) and verified that they are ISG15-conjugated in IFN-β-treated cells. The biological consequences of ISG15 modification or its association with these proteins is unclear. Previous immunofluorescence and biochemical studies have shown that ISG15 and its conjugates can be located on the cytoskeleton (31–33). One possible mechanism could be to move IFN-induced proteins from their normal location to the cytoskeleton, where they exert their antiviral activity by inhibiting either the trafficking of viral proteins or the exocytosis of viral particles.

While we were studying the role of HERC5 in ISG15 conjugation system, two groups independently reported using a proteomic approach to identify ISG15-modified proteins from HeLa cells and U937- or UBP43-deficient mouse embryonic fibroblasts (34, 35). Of 171 candidates identified in our study, only 61 proteins were found in the lists reported by the other two groups, and thus the rest of candidates represent novel substrates for ISG15 modification. Several proteins identified in our screening but not in the other two groups were examined, and they were found to be conjugated with ISG15. These proteins are 14-3-3ζ, destrin, peroxiredoxin VI, and EBNA2 coactivator.

In conclusion, we have identified a novel factor involved in ISGylation, HERC5, which acts as an ISG15 E3 protein ligase. This activity is strictly dependent on a conserved Cys residue at position 994 within the HECT domain. We also report the identification of both constitutively and IFN-induced cellular proteins that are substrates for modification by ISG15. The ISGylation of the identified protein targets was observed after type I IFN treatment or in the presence of HERC5 overexpression.

Materials and Methods

Generation of FLAG–ISG15, Myc–ISG15, UbcH8, Ube1L, and HERC5 Expression Constructs.

cDNA encoding for ISG15 was amplified from a human splenic cDNA library purchased from CLONTECH. Either two copies of FLAG tag or three copies of Myc tag were inserted at the N terminus of ISG15 and subcloned into pQXIX expression vector (CLONTECH). UbcH8 was also cloned by PCR from the human splenic cDNA library and was further subcloned into pLNCX2 expression vector (CLONTECH). Ube1L cDNA was a gift from E. Dmitrovsky (Dartmouth Medical School, Hanover, NH). Its cDNA was amplified by PCR and subcloned into pQXIX expression vector (CLONTECH). cDNA for HERC5 was kindly provided by Motoaki Ohtsubo (Kurume University, Fukuoka-ken, Japan), and an insertion of a nucleotide A at position 437 in HERC5 was deleted by site-directed mutagenesis.

Generation of A549 Cells Stably Expressing FLAG–ISG15.

A549 cells were transfected with expression construct encoding FLAG–ISG15 by Lipofectamine 2000 (Invitrogen). Cells were selected by using 500 μg/ml G418 and were further screened for FLAG–ISG15 expression by immunoblotting with anti-FLAG antibody.

Purification of ISG15 Associated and/or Modified Proteins.

A549 cells stably expressing FLAG–ISG15 (5 × 109) were treated with 500 units/ml IFN-β. Cells were lysed in 200 ml of 1% Triton X-100 in 10 mM Tris and 150 mM NaCl (pH 7.4) (TBST) on ice for 1 h. After removing nuclei and cell debris by centrifugation, crude lysate was first subjected to a mouse IgG-conjugated agarose column (Sigma) followed by an anti-FLAG M2-conjugated agarose column (Sigma) at 4°C, respectively. ISG15-associated and/or -modified proteins were eluted with 3.5 M MgCl2 after washing extensively with 1% TBST buffer. Portions of the fractions collected were separated by SDS/PAGE and were further detected by using an anti-FLAG antibody. Fractions containing FLAG–ISG15 were pooled and concentrated by using Centricon (Amicon).

MS Analysis.

MS analysis was performed in-house at the proteomic facility of the Genome Institute of Singapore. In brief, the samples were reduced and alkylated with iodoacetamide, digested with trypsin, and subjected to LC-MS/MS analysis on an LCQ Deca Plus Ion-trap mass spectrometer. Peptide masses were queried against entries in the International Protein Index human database by using mascot (Matrix Science). For a protein to be considered as a “hit,” a minimum of two matching peptides was required.

Verification of ISG15 Target Proteins.

HeLa cells were plated on 100-mm dishes (2.2 × 106) and transfected by using Lipofectamine 2000. After 4 h, dishes were washed and replaced with complete DMEM-H media either with or without IFN-β (500 units/ml). The cells were harvested after 48 h. Total lysates were extracted for Western blotting or immunoprecipitation with anti-FLAG antibody. The sequences of siRNAs and antibodies used can be found in Fig. 10. For more information, see Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Sukjo Kang, P. A. Macary, and Stephen Ogg for comments on the manuscript.

Abbreviations

siRNAsmall interfering RNA
shRNAshort hairpin RNA
ISGIFN-stimulated gene
RLDRCC1-like domain
HAhemagglutinin.

Footnotes

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

References

1. Decker T., Muller M., Stockinger S. Nat. Rev. Immunol. 2005;5:675–687. [PubMed] [Google Scholar]
2. Perry A. K., Chen G., Zheng D., Tang H., Cheng G. Cell Res. 2005;15:407–422. [PubMed] [Google Scholar]
3. Smith P. L., Lombardi G., Foster G. R. Mol. Immunol. 2005;42:869–877. [PubMed] [Google Scholar]
4. Levy D. E., Darnell J. E., Jr. Nat. Rev. Mol. Cell Biol. 2002;3:651–662. [PubMed] [Google Scholar]
5. Stark G. R., Kerr I. M., Williams B. R., Silverman R. H., Schreiber R. D. Annu. Rev. Biochem. 1998;67:227–264. [PubMed] [Google Scholar]
6. Haas A. L., Ahrens P., Bright P. M., Ankel H. J. Biol. Chem. 1987;262:11315–11323. [PubMed] [Google Scholar]
7. Korant B. D., Blomstrom D. C., Jonak G. J., Knight E., Jr. J. Biol. Chem. 1984;259:14835–14839. [PubMed] [Google Scholar]
8. Nicholl M. J., Robinson L. H., Preston C. M. J. Gen. Virol. 2000;81:2215–2218. [PubMed] [Google Scholar]
9. Kim M. J., Latham A. G., Krug R. M. Proc. Natl. Acad. Sci. USA. 2002;99:10096–10101. [PMC free article] [PubMed] [Google Scholar]
10. Ritchie K. J., Malakhov M. P., Hetherington C. J., Zhou L., Little M. T., Malakhova O. A., Sipe J. C., Orkin S. H., Zhang D. E. Genes Dev. 2002;16:2207–2212. [PMC free article] [PubMed] [Google Scholar]
11. Doyle S., Vaidya S., O’Connell R., Dadgostar H., Dempsey P., Wu T., Rao G., Sun R., Haberland M., Modlin R., Cheng G. Immunity. 2002;17:251–263. [PubMed] [Google Scholar]
12. Ritchie K. J., Zhang D. E. Semin. Cell Dev. Biol. 2004;15:237–246. [PubMed] [Google Scholar]
13. Blomstrom D. C., Fahey D., Kutny R., Korant B. D., Knight E., Jr. J. Biol. Chem. 1986;261:8811–8816. [PubMed] [Google Scholar]
14. Narasimhan J., Wang M., Fu Z., Klein J. M., Haas A. L., Kim J. J. J. Biol. Chem. 2005;280:27356–27365. [PubMed] [Google Scholar]
15. Huang D. T., Walden H., Duda D., Schulman B. A. Oncogene. 2004;23:1958–1971. [PubMed] [Google Scholar]
16. Loeb K. R., Haas A. L. J. Biol. Chem. 1992;267:7806–7813. [PubMed] [Google Scholar]
17. Dao C. T., Zhang D. E. Front. Biosci. 2005;10:2701–2722. [PubMed] [Google Scholar]
18. Yuan W., Krug R. M. EMBO J. 2001;20:362–371. [PMC free article] [PubMed] [Google Scholar]
19. Zhao C., Beaudenon S. L., Kelley M. L., Waddell M. B., Yuan W., Schulman B. A., Huibregtse J. M., Krug R. M. Proc. Natl. Acad. Sci. USA. 2004;101:7578–7582. [PMC free article] [PubMed] [Google Scholar]
20. Kim K. I., Giannakopoulos N. V., Virgin H. W., Zhang D. E. Mol. Cell. Biol. 2004;24:9592–9600. [PMC free article] [PubMed] [Google Scholar]
21. Nyman T. A., Matikainen S., Sareneva T., Julkunen I., Kalkkinen N. Eur. J. Biochem. 2000;267:4011–4019. [PubMed] [Google Scholar]
22. Pickart C. M., Eddins M. J. Biochim. Biophys. Acta. 2004;1695:55–72. [PubMed] [Google Scholar]
23. Scheffner M., Huibregtse J. M., Vierstra R. D., Howley P. M. Cell. 1993;75:495–505. [PubMed] [Google Scholar]
24. Huibregtse J. M., Scheffner M., Beaudenon S., Howley P. M. Proc. Natl. Acad. Sci. USA. 1995;92:5249. [PMC free article] [PubMed] [Google Scholar]
25. Joazeiro C. A., Weissman A. M. Cell. 2000;102:549–552. [PubMed] [Google Scholar]
26. Mitsui K., Nakanishi M., Ohtsuka S., Norwood T. H., Okabayashi K., Miyamoto C., Tanaka K., Yoshimura A., Ohtsubo M. Biochem. Biophys. Res. Commun. 1999;266:115–122. [PubMed] [Google Scholar]
27. Garcia-Gonzalo F. R., Rosa J. L. Cell. Mol. Life Sci. 2005;62:1826–1838. [PMC free article] [PubMed] [Google Scholar]
28. Hochrainer K., Mayer H., Baranyi U., Binder B., Lipp J., Kroismayr R. Genomics. 2005;85:153–164. [PubMed] [Google Scholar]
29. Mueller D. L. Nat. Immunol. 2004;5:883–890. [PubMed] [Google Scholar]
30. Dastur A., Beaudenon S., Kelley M., Krug R. M., Huibregtse J. M. J. Biol. Chem. 2006;281:4334–4338. [PubMed] [Google Scholar]
31. Austin K. J., Carr A. L., Pru J. K., Hearne C. E., George E. L., Belden E. L., Hansen T. R. Endocrinology. 2004;145:967–975. [PubMed] [Google Scholar]
32. Loeb K. R., Haas A. L. Mol. Cell. Biol. 1994;14:8408–8419. [PMC free article] [PubMed] [Google Scholar]
33. Bebington C., Bell S. C., Doherty F. J., Fazleabas A. T., Fleming S. D. Biol. Reprod. 1999;60:920–928. [PubMed] [Google Scholar]
34. Zhao C., Denison C., Huibregtse J. M., Gygi S., Krug R. M. Proc. Natl. Acad. Sci. USA. 2005;102:10200–10205. [PMC free article] [PubMed] [Google Scholar]
35. Giannakopoulos N. V., Luo J. K., Papov V., Zou W., Lenschow D. J., Jacobs B. S., Borden E. C., Li J., Virgin H. W., Zhang D. E. Biochem. Biophys. Res. Commun. 2005;336:496–506. [PubMed] [Google Scholar]
Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
-