HERC5 is an IFN-induced HECT-type E3 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*‡§¶

*Immunology and Virology Laboratory and †Proteomics Laboratory, Genome Institute of Singapore, 60 Biopolis Street, #02-01 Genome, Singapore 138672; ‡Department of Microbiology and Immunology, Yong Loo Lin School of Medicine, Block MD4, 5 Science Drive 2, Singapore 117597; and §Immunology Program, National University of Singapore, Office of Life Sciences Satellite Laboratory, Defense Medical and Environmental Research Institute Building, #03-01, 27 Medical Drive, Singapore 117510

Edited by Peter Cresswell, Yale University School of Medicine, New Haven, CT, and approved May 26, 2006 (received for review January 16, 2006) Type I IFNs induce the expression of IFN-stimulated 15 (ISG15) total level of IFN-induced conjugation of ISG15 to cellular and its conjugation to cellular targets. ISGylation is a multistep (19, 20). process involving IFN-inducible Ube1L, UbcH8, and a yet-to-be In the and ubiquitin-like proteins (ublps) system, E3 identified E3 ligase. Here we report the identification of an IFN- enzymes play a critical role in transferring ubiquitin or ublps such induced HECT-type E3 protein ligase, HERC5͞Ceb1, which mediates as SUMO from the E2-conjugating enzyme to a specific substrate. ISGylation. We also defined a number of proteins modified by The substrate protein interacts directly with a specific domain in the ISG15 after IFN triggering or HERC5 overexpression. A reduction in E3 enzymes or through an adaptor associated with the E3 enzyme endogenous HERC5 by small interfering RNA inhibition blocks the (22). E3 enzymes are known to be divided into two groups: HECT IFN-induced ISG15 conjugation. Conversely, HERC5 coexpression (homologous to E6-AP C terminus) and RING (really interesting with Ube1L and UbcH8 induces the ISG15 conjugation in vivo new ) proteins (22–25). Whereas RING-type proteins transfer independent of IFN stimulation. A targeted substitution of Cys-994 ubiquitin directly from an E2 to a target, the HECT-type protein to Ala in the HECT domain of HERC5 completely abrogates its E3 forms a thioester bond with ubiquitin or ublps by its active cysteine protein ligase activity. Therefore, this study demonstrates that residue before transferring it to a substrate (25). The identity of the HERC5͞Ceb1 is involved in the conjugation of ISG15 to cellular E3 enzyme(s) in the ISG15 conjugation system has not been proteins. resolved. Here we report the identification of target proteins conjugated ISG15 ͉ Ceb1 ͉ innate immunity ͉ antiviral proteins 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 li- ype I IFNs (IFN-␣͞␤) play an essential role in both innate gase, HERC5, which itself is also a target for modification by antiviral and adaptive immune responses and are rapidly pro- T ISG15. HERC5 catalyzes ISGylation by means of a catalytic duced in response to microbial infection (1–3). They exert signals cysteine residue at position 994 in the HECT domain. Moreover, through the activation of the Janus kinase–signal transducer and HERC5 is found to be sufficient for inducing ISGylation in the activator of transcription pathway that mediates rapid induction of absence of IFN. Knock-down of HERC5 by small interfering IFN-stimulated genes (ISGs) (4, 5). ISG15 is one of the most RNA (siRNA) results in the inhibition of IFN-induced ISG15 strongly induced genes after IFN treatment (6, 7) and is also conjugation. Thus, HERC5 is an IFN-induced E3 protein ligase significantly induced by viral infection (8, 9) and LPS treatment (10, that mediates ISGylation. 11). The ISG15 protein starts with two ubiquitin-related domains that have Ϸ27% sequence identity to ubiquitin and terminate in a Results 152 157 conserved LRLRGG ubiquitin C-terminal motif. This study Identification and Purification of FLAG–ISG15-Associated and͞or suggests that ISG15 could act in a similar way to ubiquitin and other -Modified Proteins. To search for potential E3 enzyme(s) as well as ubiquitin-like proteins such as SUMO by forming an isopeptide proteins that were physically associated or covalently conjugated bond with cellular proteins (6, 12, 13). The crystal structure of with ISG15, we generated A549 cell lines stably expressing ISG15 ISG15 revealed that ISG15 consists of two domains with ubiquitin- with two copies of FLAG tag (Fig. 1A). The FLAG tags were like folds joined by a linker sequence (14). inserted at the N terminus of ISG15 because of the involvement of Conjugation of ISG15 to cellular proteins occurs in a parallel but the conserved C terminus diglycine motif in conjugation with IMMUNOLOGY distinct mechanism to that of ubiquitin (15–17). The E1 enzyme for cellular proteins (Fig. 1A). The FLAG–ISG15 retains its conjuga- ISG15, Ube1L, is a single-subunit enzyme and is identified in vitro tion activity to cellular proteins after 48 h of IFN-␤ treatment as by its ability to catalyze the formation of a thioester bond between compared with untreated control (Fig. 7A, which is published as ISG15 and Ube1L (17, 18). The Ube1L protein is highly similar to supporting information on the PNAS web site). These data indicate the E1 enzyme for ubiquitin at the protein level. However, this that FLAG tags inserted at the N terminus of ISG15 do not disrupt protein does not form a conjugate with ubiquitin, indicating that the conjugation to cellular proteins after IFN-␤ treatment. Ube1L is an E1 enzyme for the ISG15 conjugation system (ISGy- We next carried out large-scale isolation of ISG15-associated͞ lation). 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 ISGy- Conflict of interest statement: No conflicts declared. lation might be important for protecting cells from viral infection. This paper was submitted directly (Track II) to the PNAS office. Two groups recently found that a member of the ubiquitin Freely available online through the PNAS open access option. E2-conjugating enzyme family, UbcH8, is involved in the ISGyla- Abbreviations: siRNA, small interfering RNA; shRNA, short hairpin RNA; ISG, IFN-stimulated tion (19, 20). Like ISG15 and Ube1L, the expression of UbcH8 is gene; RLD, RCC1-like domain; HA, hemagglutinin. also induced by IFN (21). The suppression of UbcH8 protein ¶To whom correspondence should be addressed. E-mail: [email protected]. expression by RNA interference is shown to dramatically inhibit the © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600397103 PNAS ͉ July 11, 2006 ͉ vol. 103 ͉ no. 28 ͉ 10735–10740 Downloaded by guest on October 2, 2021 Fig. 1. Identification and purification of ISG15-associated and͞or -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 Ϸ15 kDa. Indeed, ISG15 protein runs as 15 kDa in SDS͞PAGE. (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-␤ for 48 h. Total lysates were immunoprecipitated with anti-FLAG M2-conjugated agarose and immunoblotted with antibodies against destrin, cofilin, enolase, and GAPDH.

conjugated proteins by anti-FLAG immunoaffinity purification information on the PNAS web site, HERC5 was represented by five (Fig. 7B). Control A549 fractions were also analyzed to identify individual peptides from tandem MS sequencing and was not found nonspecific, copurified proteins and hence eliminate false positives. in the control sample, indicating that this protein is specifically A total of 174 unique candidate proteins, not found in control copurified with FLAG–ISG15. fractions, and represented by at least two peptides in the tandem The primary sequence of HERC5 revealed the presence of MS analysis, were identified. The ISG15 target proteins that RCC1-like (regulator of condensation-1) and HECT fulfilled these stringent criteria are listed in Table 1, which is domains that span residues 209–258 and 676-1024, respectively (Fig. published as supporting information on the PNAS web site. 8A) (26). The HECT domain was previously demonstrated to Twenty-six target proteins were selected for further validation of interact with a ubiquitin-conjugating enzyme (23). Within the their conjugation or association with ISG15. Twenty-four of 26 HECT domain of HERC5, a conserved cysteine residue residing in putative ISG15 target proteins were found to form conjugates with all known HECT-type protein ligases was also found and located at ISG15 after IFN-␤ treatment (Table 1, bold). Two other ISG15- position 994 (Fig. 8B) (26). Because HERC5͞Ceb1 possesses all of copurified proteins, and pICln, were not directly conjugated the unique structural features of a mammalian E3 protein ligase, we to ISG15 but formed a specific, noncovalent interaction (data not hypothesized that HERC5 functions as an E3 protein ligase in shown). Of the proteins that were substrates for ISG15 conjugation, ISGylation. a number, such as destrin and cofilin, were found to conjugate with Because HERC5 is identified by the pulling down of ISGylated a single ISG15 as they migrated at the expected molecular masses proteins and ISG15-associated proteins, we first determined for destrin*FLAG–ISG15 (Mr Ϸ 37 kDa) (Fig. 1B, lane 2) and whether ISG15 binds HERC5 or forms covalent conjugated species cofilin*FLAG–ISG15 (Mr Ϸ 37 kDa) (Fig. 1C, lane 2). On the other with HERC5. HeLa cells were cotransfected with FLAG–HERC5 hand, some proteins, such as enolase and GAPDH, existed as and Myc–ISG15 (Fig. 2A, lanes 1–4), and cells were treated with multiply conjugated species that were possibly modified by one or (Fig. 2A, lanes 3 and 4) or without (Fig. 2A, lanes 1 and 2) IFN-␤. more ISG15s (Fig. 1 D and E, lane 2). At least two isoforms of We performed either anti-FLAG (Fig. 2A, lanes 2 and 4) or control enolase-conjugated species were observed, migrating at the posi- (Fig. 2A, lanes 1 and 3) immunoprecipitation followed by anti-Myc tions expected for one and three ISG15s (Fig. 1D, lane 2). Similar or anti-FLAG Western blotting to determine whether higher- to enolase, GAPDH also displayed a number of conjugated species molecular-weight forms of HERC5 or free forms of ISG15 could that migrated at the positions expect for one and two ISG15s in be detected. As shown in Fig. 2A, we saw multiple species of high SDS͞PAGE (Fig. 1E, lane 2). In addition to conjugation, we also molecular forms of HERC5 in IFN-␤-treated cells expressing both observed noncovalent association between ISG15 and cellular FLAG–HERC5 and Myc–ISG15 by anti-Myc and anti-FLAG substrates (Fig. 1 B–E, shown by arrowhead). Thus, we have Western blotting, respectively. Moreover, we saw free forms of identified a large number of cellular proteins that form either single ISG15 copurified with HERC5 in both untreated and IFN-␤- or multiple conjugation and noncovalent interactions with ISG15. treated cells (Fig. 2A Left). These data indicate that HERC5 physically associates with ISG15 and itself is a target for modifica- HERC5͞Ceb1 Is Copurified with ISG15, and Its Expression Is Regulated tion by ISG15. by Type I IFN. Conjugation of ISG15 with cellular proteins is We then sought to examine whether type I IFN induces HERC5 catalyzed by the E1 activating enzyme Ube1L and the E2- mRNA expression in both HeLa and A549 cell lines because the conjugating enzyme UbcH8 (17). Thus far, no E3 enzyme(s) has expression of Ube1L and UbcH8 has previously been shown to be been identified. In ubiquitin system, E3 enzymes, with either a induced by IFN-␤ (21). Both HeLa and A549 cells were treated with HECT domain or a RING domain, play a critical role in recruiting IFN for 6, 12, 24, and 48 h. As shown in Fig. 2B, we detected basal the ubiquitin-loaded E2, recognizing specific substrates and facili- levels of HERC5 mRNA in both untreated HeLa (Left) and A549 tating the transfer of the ubiquitin from the E2 to the lysine residue (Right) cells. Upon IFN-␤ stimulation, HERC5 mRNA was rapidly in the substrates (22). Among all ISG15-associated͞conjugated induced in both cell lines after 6 h (Fig. 2B). At 12 h there were candidate proteins, we noticed that a protein known as HERC5 (or Ϸ30-fold increments in HERC5 mRNA expression levels compared Ceb1) contains a HECT-type domain at its C terminus (Fig. 8, with their respectively untreated controls for both HeLa and A549 which is published as supporting information on the PNAS web site) cells (Fig. 2B). The mRNA for HERC5 was continuously expressed (26). As shown in Table 2, which is published as supporting at high levels at 24 and 48 h after IFN-␤ treatment as compared with

10736 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600397103 Wong et al. Downloaded by guest on October 2, 2021 siRNAs against HERC5. After 48 h of IFN-␤ treatment, total lysates were made and separated in SDS͞PAGE. ISG15 conjugates were detected by immunoblotting with an antibody against endog- enous 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-␤ 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-␤ is necessary for catalyzing ISGylation in A549 cells. Next we sought to test whether the inducibility of endogenous ISG15 conjugates by IFN-␤ 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 gen- erated. 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-␤ 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-␤ 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 conju- gates after 48 h of IFN-␤ treatment (Fig. 3E). Taken together, these results indicate that HERC5 plays an essential role in catalyzing Fig. 2. HERC5 is an IFN-inducible protein and is covalently modified with IFN-␤-induced ISGylation in both HeLa and A549 cells. ISG15. (A) HERC5 interacts with ISG15 and forms conjugated species with ISG15. HeLa cells were cotransfected with FLAG–HERC5 and Myc–ISG15, and Coexpression of HERC5 with Ube1L and UbcH8 Restores ISG15 Conju- cells were treated (lanes 3 and 4) or not treated (lanes 1 and 2) with IFN-␤. Total lysates were prepared and immunoprecipitated with either control (lanes 1 gates in Vivo Without Type I IFN for Induction. Because HERC5 and 3) or FLAG (lanes 2 and 4) antibody and then separated on SDS͞PAGE. The expression is induced by IFN, we tested whether overexpression of protein complexes were detected with either anti-Myc (Left) or anti-FLAG HERC5 in combination with Ube1L and UbcH8 would lead to (Right) antibody. (B) Kinetics of IFN-induced HERC5 mRNA expression in HeLa ISGylation in the absence of IFN-␤. HeLa cells transfected with (Left) and A549 (Right) cells. HeLa and A549 cells were either untreated or either Ube1L (Fig. 4A, lane 2) or UbcH8 (Fig. 4A, lane 3) expressed treated with IFN-␤ for 6, 12, 24, and 48 h. Total RNAs were isolated, and no ISG15 conjugates. A partial expression of ISG15 conjugates was real-time PCR was performed with HERC5-specific primers. (C) Induction of observed in HeLa cells transfected with Ube1L and UbcH8 (Fig. ISG15 conjugation in HeLa (Left) and A549 (Right) cells after IFN-␤ treatment. 4A, lane 4), which may have been because of a low level of HERC5 The unstimulated and IFN-stimulated HeLa and A549 cells were harvested at expression in HeLa cells (refer to RNA work in Fig. 2B Left). When 6, 12, 24, and 48 h. Total lysates were separated in SDS͞PAGE and immuno- blotted with anti-ISG15 antibody. 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 overex- the controls (Fig. 2B). We also measured the induction of ISG15 pression is sufficient to drive ISGylation. conjugates in both HeLa (Fig. 2C Left) and A549 (Fig. 2C Right) Next we examined a number of proteins known to be ISGy- ␤ cells upon IFN-␤ treatment. In contrast to the kinetics of HERC5 lated after IFN- treatment, for conjugation with ISG15 in HeLa

mRNA expression, ISG15 protein conjugates were only detected in cells overexpressing HERC5. HeLa cells were transfected with IMMUNOLOGY both HeLa and A549 cells at 24 h after IFN-␤ treatment (Fig. 2C, FLAG–ISG15 alone (Fig. 4 B–I, lane 1), FLAG–ISG15 plus lane 4) and were continually observed at 48 h (Fig. 2C, lane 5). vectors expressing Ube1L and UbcH8 (Fig. 4 B–I, lane 2), or B I Together, these data indicated that HERC5 is an IFN-inducible HERC5, Ube1L, and UbcH8 (Fig. 4 – , lane 3). Cells were harvested after 48 h. FLAG–ISG15 conjugates were immuno- protein. Also, the strong induction of HERC5 mRNA expression by precipitated with anti-FLAG antibody. The protein complexes IFN-␤ before ISGylation is paralleled to the observations made for were separated in SDS͞PAGE and detected with specific anti- Ube1L and UbcH8. Thus, we hypothesized that HERC5 could bodies. All of the target proteins examined were found to be function as a protein ligase in concert with Ube1L and UbcH8 to covalently conjugated with ISG15 when HERC5 is overex- ␤ promote ISGylation upon IFN- treatment. pressed (Fig. 4 B–I, lanes 3). Three target proteins, destrin, enolase, and GAPDH, were observed to have low levels of Depletion of Endogenous HERC5 Inhibits ISGylation upon Type I IFN conjugation in HeLa cells transfected with Ube1L and UbcH8 Treatment. To investigate the role of HERC5 in ISGylation, we used alone (Fig. 4 B, D, and E, lane 2). However, their levels of siRNA technology to suppress the expression of endogenous conjugation were dramatically enhanced in cells transfected with HERC5 mRNA in A549 cells. Two small siRNAs, namely siRNA– HERC5, Ube1L, and UbcH8 (Fig. 4 B, D, and E, lane 3). HERC5-I and siRNA–HERC5-II, which target nucleotide se- Interestingly, the presence of multiple conjugated species for quences 536–554 bp and 715–735 bp, respectively, in HERC5 were both enolase and GAPDH were also observed after HERC5 used. A549 cells were transfected with either control siRNA or overexpression, indicating that HERC5 is essential for both

Wong et al. PNAS ͉ July 11, 2006 ͉ vol. 103 ͉ no. 28 ͉ 10737 Downloaded by guest on October 2, 2021 Fig. 3. Depletion of HERC5 inhibits the induction of ISG15 conjugation by IFN-␤.(A) HERC5 depletion by siRNAs inhibits ISG15 conjugation induced by IFN-␤ 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-␤ for another 48 h. Total lysates were harvested and separated in SDS͞PAGE. 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-␤ 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-␤ 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-␤-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-␤-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 ␮g͞ml puromycin. Cells were treated with IFN-␤ for 48 h, and ISG15 conjugates were analyzed by immunoblotting with anti-ISG15 antibody.

single and multiple ISGylation in target proteins (Fig. 4 D and intermediate bond with ubiquitin before transferring it to the E, lane 3). In contrast to enolase and GAPDH, the other five substrates (Fig. 8B). To further demonstrate that HERC5 is an target proteins tested (cofilin, , Hsc70, peroxiredoxin I, E3 protein ligase for ISGylation, the putative active cysteine and peroxiredoxin VI) displayed no detectable ISGylation in residue (C994), based on conservation with other HECT do- HeLa cells transfected with only Ube1L and UbcH8 (Fig. 4 C main-containing E3s, was mutated to an alanine residue by and F–I, lane 2). These proteins all exhibited significant levels of site-specific mutagenesis. HeLa cells were cotransfected with ISG15 conjugation in the HeLa cells overexpressed with HERC5 Myc–ISG15 and either wild-type FLAG–HERC5 (Fig. 5, lane 4) (Fig. 4 C and F–I, lane 3). Together, these data indicate that or mutant FLAG–HERC5 (Fig. 5, lane 5). This point HERC5 is an E3 protein ligase involved in ISGylation. Both single and multiple conjugations in target proteins require the in the cysteine residue (C994) completely abolishes its E3 activity expression of HERC5. Once expressed, the enzymatic activity of in ISGylation (Fig. 5 Upper, compare lanes 4 and 5). The HERC5 is not dependent on type I IFNs. expression levels of wild-type and mutant HERC5 proteins were found to be comparable (Fig. 5 Lower). These data demonstrate Cys-994 Within the HECT Domain of HERC5 Is Essential for Its Protein that HERC5 acts as an E3 protein ligase in ISGylation by means Ligase Activity in ISGylation. All HECT-type ubiquitin protein of a conserved cysteine residue, C994, found in the HECT ligases contain a conserved active cysteine residue that forms an domain.

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 SDS͞PAGE 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).

10738 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0600397103 Wong et al. Downloaded by guest on October 2, 2021 ISGylation by recruiting cellular substrates for modification by ISG15, which is also found to be associated with HERC5 (Fig. 2A). Discussion HERC5 Is an IFN-Inducible Protein Ligase for ISG15 Conjugation Sys- tem. 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-␤. 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 mem- bers, 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 Fig. 5. Conserved Cys-994 within the HECT domain of HERC5 is essential for the induction of ISG15 conjugation induced by IFN. Second, its ligase activity in ISGylation. Cys-994 within the HECT domain of HERC5 was HERC5 coexpression with the Ube1L and UbcH8 suffices to mutated to alanine residue. HeLa cells were transfected with vectors express- ing 3Myc–ISG15 (lane 2); 3Myc–ISG15, Ube1L, and UbcH8 (lane 3); 3Myc–ISG15, mediate ISG15 conjugation in vivo even if in the absence of IFN Ube1L, UbcH8, and wild-type FLAG–HERC5 (lane 4); and 3Myc–ISG15, Ube1L, treatment. Third, a mutation of a conserved cysteine residue in the UbcH8, and FLAG–HERC5–C994A (lane 5). Forty-eight hours after transfection, HECT domain of HERC5 to an alanine residue abolished its cell lysates were collected, and Myc–ISG15 conjugates were detected by protein ligase activity in vivo. This result is consistent with the immunoprecipitation followed by immunoblotting with anti-Myc antibody hypothesis that C994 is important for transferring ISG15 from (Upper). The expression of wild-type and mutant HERC5 was detected with UbcH8 to specific target proteins. Fourth, HERC5 interacts with anti-FLAG antibody (Lower). 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 HERC5 Interacts with ISGylated Substrates, HSC70, and Thioredoxin could act as an E3 protein ligase for ISGylation. In addition, we find Reductase. To further support that HERC5 is a protein ligase for that knock-down of Ube1, E1 enzyme for the ubiquitination system, ISG15, we next tested whether HERC5 interacts with ISGylated has no affect on the ISGylation, and this result rules out the substrates, Hsc70, and thioredoxin reductase. HeLa cells were possibility that HERC5 acts an E3 enzyme for ubiquitination by transfected with FLAG–HERC5 (Fig. 6, lane 3), ISGylated sub- activating an uncharacterized E3 enzyme for ISGylation and in- strate (Fig. 6, lane 2), FLAG–HERC5 plus HA-Hsc70 (Fig. 6A, ducing ISGylation (Fig. 10, which is published as supporting infor- lane 4) or HA–thioredoxin reductase (Fig. 6B, lane 4). Cell extracts mation on the PNAS web site). The finding that HERC5 is involved were immunoprecipitated with an anti-FLAG monoclonal anti- in ISGylation in this study was also demonstrated by an independent body. The immunoprecipitates were then subjected to SDS͞PAGE group while this article was under review (30). One of the chal- and immunoblotted with antibodies against FLAG or hemaggluti- lenging tasks is to express HERC5 for promotion of ISGylation in nin (HA). As shown in Fig. 6, the antibody against FLAG precip- vitro; however, we and Dastur et al. (30) had some difficulty itated FLAG–HERC5 in cells expressing FLAG–HERC5 (Fig. 6 expressing either the full-length or the HECT domain of HERC5 Middle, lanes 3 and 4), but not in the control cells (Fig. 6 Middle, in the bacterial system, and this issue remains to be resolved. lanes 1 and 2). The FLAG antibody also precipitated Hsc70 (Fig. The N-terminal region of HERC5 possesses at least one RLD. 6A Top, lane 4) or thioredoxin reductase (Fig. 6B Top, lane 4). The RLDs have been shown to interact with several proteins, and it is expression of cellular substrates was the same by immunoblotting possible that the RLDs serve as the regions for recruiting specific cell lysates with an HA antibody (Fig. 6 Bottom, lanes 2 and 4). protein substrates. In addition, the middle region, between RLDs These results suggest that HERC5 acts as an E3 protein ligase for 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 IMMUNOLOGY 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 Fig. 6. HERC5 interacts with ISGylated substrates, Hsc70, and thioredoxin conjugation pathway, we are highly confident that our large data set reductase. (A) HeLa cells were transfected with HA-Hsc70 (lane 2), FLAG– represents bona fide substrates for ISG15 in vivo. HERC5 (lane 3), or FLAG–HERC5 plus HA-Hsc70 (lane 4). Total lysates were Eight putative IFN-inducible ISG15 targets (MxA, IFIT1, signal made and immunoprecipitated with anti-FLAG agarose. The immunocom- plexes were separated and immunoblotted with anti-HA (Top) for detection transducer and activator of transcription 1, Ube1L, tryptophanyl of Hsc70 or anti-FLAG (Middle) for HERC5. The total amount of Hsc70 was tRNA synthetase, TRIM21, gelsolin, and HERC5) are identified confirmed by immunoblotting of cell lysate with anti-HA antibody (Bottom). (4, 5). We chose four of the IFN-induced proteins (MxA, IFIT1, (B) Same as above, except that the HA-tagged form of thioredoxin reductase signal transducer and activator of transcription 1, and tryptophanyl (HA-THR) was used in the transfection. tRNA synthetase) and verified that they are ISG15-conjugated in

Wong et al. PNAS ͉ July 11, 2006 ͉ vol. 103 ͉ no. 28 ͉ 10739 Downloaded by guest on October 2, 2021 IFN-␤-treated cells. The biological consequences of ISG15 modi- Generation of A549 Cells Stably Expressing FLAG–ISG15. A549 cells fication or its association with these proteins is unclear. Previous were transfected with expression construct encoding FLAG–ISG15 immunofluorescence and biochemical studies have shown that by Lipofectamine 2000 (Invitrogen). Cells were selected by using ISG15 and its conjugates can be located on the cytoskeleton 500 ␮g͞ml G418 and were further screened for FLAG–ISG15 (31–33). One possible mechanism could be to move IFN-induced expression by immunoblotting with anti-FLAG antibody. proteins from their normal location to the cytoskeleton, where they exert their antiviral activity by inhibiting either the trafficking of Purification of ISG15 Associated and͞or Modified Proteins. A549 cells viral proteins or the exocytosis of viral particles. stably expressing FLAG–ISG15 (5 ϫ 109) were treated with 500 While we were studying the role of HERC5 in ISG15 conjugation units͞ml IFN-␤. Cells were lysed in 200 ml of 1% Triton X-100 in system, two groups independently reported using a proteomic 10 mM Tris and 150 mM NaCl (pH 7.4) (TBST) on ice for 1 h. After approach to identify ISG15-modified proteins from HeLa cells and removing nuclei and cell debris by centrifugation, crude lysate was U937- or UBP43-deficient mouse embryonic fibroblasts (34, 35). Of first subjected to a mouse IgG-conjugated agarose column (Sigma) 171 candidates identified in our study, only 61 proteins were found followed by an anti-FLAG M2-conjugated agarose column (Sigma) in the lists reported by the other two groups, and thus the rest of at 4°C, respectively. ISG15-associated and͞or -modified proteins candidates represent novel substrates for ISG15 modification. Sev- were eluted with 3.5 M MgCl2 after washing extensively with 1% eral proteins identified in our screening but not in the other two TBST buffer. Portions of the fractions collected were separated by groups were examined, and they were found to be conjugated with SDS͞PAGE and were further detected by using an anti-FLAG ISG15. These proteins are 14-3-3␨, destrin, peroxiredoxin VI, and antibody. Fractions containing FLAG–ISG15 were pooled and EBNA2 coactivator. concentrated by using Centricon (Amicon). 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 MS Analysis. MS analysis was performed in-house at the proteomic 994 within the HECT domain. We also report the identification of facility of the Genome Institute of Singapore. In brief, the samples were reduced and alkylated with iodoacetamide, digested with both constitutively and IFN-induced cellular proteins that are ͞ substrates for modification by ISG15. The ISGylation of the iden- trypsin, and subjected to LC-MS MS analysis on an LCQ Deca Plus tified protein targets was observed after type I IFN treatment or in Ion-trap mass spectrometer. Peptide masses were queried against the presence of HERC5 overexpression. entries in the International Protein Index human database by using MASCOT (Matrix Science). For a protein to be considered as a ‘‘hit,’’ Materials and Methods a minimum of two matching peptides was required. Generation of FLAG–ISG15, Myc–ISG15, UbcH8, Ube1L, and HERC5 Expression Constructs. cDNA encoding for ISG15 was amplified Verification of ISG15 Target Proteins. HeLa cells were plated on from a human splenic cDNA library purchased from CLONTECH. 100-mm dishes (2.2 ϫ 106) and transfected by using Lipo- Either two copies of FLAG tag or three copies of Myc tag were fectamine 2000. After 4 h, dishes were washed and replaced with inserted at the N terminus of ISG15 and subcloned into pQXIX complete DMEM-H media either with or without IFN-␤ (500 expression vector (CLONTECH). UbcH8 was also cloned by PCR units͞ml). The cells were harvested after 48 h. Total lysates were from the human splenic cDNA library and was further subcloned extracted for Western blotting or immunoprecipitation with into pLNCX2 expression vector (CLONTECH). Ube1L cDNA was anti-FLAG antibody. The sequences of siRNAs and antibodies a gift from E. Dmitrovsky (Dartmouth Medical School, Hanover, used can be found in Fig. 10. For more information, see NH). Its cDNA was amplified by PCR and subcloned into pQXIX Supporting Materials and Methods, which is published as sup- expression vector (CLONTECH). cDNA for HERC5 was kindly porting information on the PNAS web site. provided by Motoaki Ohtsubo (Kurume University, Fukuoka-ken, Japan), and an insertion of a nucleotide A at position 437 in We thank Sukjo Kang, P. A. Macary, and Stephen Ogg for comments on HERC5 was deleted by site-directed mutagenesis. the manuscript.

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