Interferon-inducible ubiquitin E2, Ubc8, is a conjugating enzyme for protein ISGylation

Abstract

Protein ISGylation is unique among ubiquitin-like conjugation systems in that the expression and conjugation processes are induced by specific stimuli, mainly via the alpha/beta interferon signaling pathway. It has been suggested that protein ISGylation plays a special role in the immune response, because of its interferon-signal dependency and its appearance only in higher eukaryotic organisms. Here, we report the identification of an ISG15-conjugating enzyme, Ubc8. Like other components of the protein ISGylation system (ISG15, UBE1L, and UBP43), Ubc8 is an interferon-inducible protein. Ubc8 clearly mediates protein ISGylation in transfection assays. The reduction of Ubc8 expression by small interfering RNA causes a decrease in protein ISGylation in HeLa cells upon interferon treatment. Neither UbcH7/UbcM4, the closest homologue of Ubc8 among known ubiquitin E2s, nor the small ubiquitin-like modifier E2 Ubc9 supports protein ISGylation. These findings strongly suggest that Ubc8 is a major ISG15-conjugating enzyme responsible for protein ISGylation upon interferon stimulation. Furthermore, we established an assay system to detect ISGylated target proteins by cotransfection of ISG15, UBE1L, and Ubc8 together with a target protein to be analyzed. This method provides an easy and effective way to identify new targets for the ISGylation system and will facilitate related studies.

FIG. 1.

Correlation between Ubc8 expression and protein ISGylation. NIH 3T3 cells (A) and primary lung fibroblasts from wild-type (WT) and STAT1^−/− mice (B) were treated with 500 U of mouse IFN-β/ml for the indicated times. Protein ISGylation and UbcM8 expression were detected by Western blotting. Molecular weight markers (in thousands) are indicated on the left side of each panel. Ponceau staining shows the relative amounts of protein in the samples. (C) Promoter regions of human genes for ISG15, UBE1L, UBP43, and UbcH8 contain ISRE (underlined). Conj., conjugate.

FIG. 2.

Interaction between ISG15 and UbcM8 in yeast two-hybrid screening. (A) Full-length ISG15 (amino acids 1 to 161) or the first ubl domain of ISG15 (amino acids 1 to 79) was used as bait for yeast two-hybrid screening to identify ISG15-interacting proteins. (B) The clone from the domain 1 screening (UbcM8 amino acids 54 to 153) was retransformed into AH109 and mated withY187 expressing lamin, p53, full-length ISG15 (FL-ISG15), ISG15-GG, or domain 1 or domain 2 of ISG15. The − and + indicate negative and positive interactions of these proteins with UbcM8, respectively.

FIG. 3.

Ubc8 mediates protein ISGylation. Plasmid constructs pCAGGS-mISG15, pCAGGS-HA-hUBE1L, pcDNA3-HA-9-27, and pFlagCMV2-UbcM8 (A), and pCAGGS-5HA-mISG15, pCAGGS-HA-hUBE1L, and pFlagCMV2-UbcM8 (B) were transfected into 293T cells as indicated on the panels. Cells were harvested 48 h after transfection, and protein extracts were analyzed on an SDS-8 to 18% PAGE gel followed by Western blotting. Free or conjugated (Conj.) forms of ISG15 were detected with rabbit anti(α)-mISG15 polyclonal antibody (A) and with α-HA antibody (B). Other protein expressions were monitored with α-HA or α-Flag antibody. Plasmids are identified by the ending letters and/or numbers of each designation. The asterisk in panel B indicates nonspecific signal from α-HA antibody.

FIG. 4.

Mutational analysis of active-site cysteine on both UBE1L and UbcM8. Plasmid constructs pCAGGS-mISG15, pCAGGS-HA-hUBE1L, pCAGGS-HA-hUBE1LC598A, pFlagCMV2-UbcM8, and pFlagCMV2-UbcM8C86A (A) and pCAGGS-mISG15, pCAGGS-HA-hUBE1L, pFlagCMV2-UbcM8, and pFlagCMV2-UbcH8 (B) were transfected into 293T cells in combinations as indicated. The expression of proteins and protein ISGylation were analyzed as described for Fig. 3. Plasmids are identified by the ending letters and numbers of each designation. Conj., conjugate.

FIG. 5.

Ubc8 but not its closest homologue, UbcH7/UbcM4, can support protein ISGylation. (A) Plasmid constructs pCAGGS-mISG15, pCAGGS-HA-hUBE1L, pFlagCMV2-UBE1, pFlagCMV2-UbcM8, pFlagCMV2-UbcM4, and pFlagCMV2-Ubc9 were transfected into 293T cells. The expression of proteins and protein ISGylation were analyzed as described for Fig. 3. (B) Increasing amounts of Flag-UBE1 (pFlagCMV2-UBE1) or HA-UBE1L (pCAGGS-HA-hUBE1L) were transfected into 293T cells together with Myc-Ub-expressing plasmid. After 40 h of transfection, cells were treated with 10 μM MG132 for 8 h. Protein ubiquitylation was analyzed by Western blotting with anti-Myc antibody. Plasmids are identified by the ending letters and numbers of each designation. Conj., conjugate.

FIG. 6.

siRNA for UbcH8 blocks IFN-induced protein ISGylation. Increasing amounts of MSCV-UbcH8 shRNA construct were cotransfected with pFlagCMV2-UbcH8, pCAGGS-mISG15, and pCAGGS-HA-hUBE1L to 293T cells. Forty-eight hours after transfection, cell extracts were subjected to Western blot analysis to examine protein expression for Flag-UbcH8 and HA-UBE1L and also to examine protein ISGylation. Plasmids are identified by the ending letters and numbers of each designation (A). HeLa cells were transfected with control MSCV vector or MSCV-UbcH8 shRNA construct. Twenty-four hours posttransfection, cells were selected with 2 μg of puromycin/ml for 24 h. Dead cells were washed out with PBS, and the remaining HeLa cells were treated with 5,000 U of IFN-α/ml. RNA and protein samples were prepared after treating with IFN for 12 and 24 h, respectively. RNA samples were analyzed by Northern blotting with an UbcH8 probe (B), and proteins were subjected to Western blotting for ISGylation (C). The asterisk in panel B indicates nonspecific signal. For 2D gel electrophoresis, HeLa extracts were prepared as described for panel C, separated on an 11-cm 2D gel until the 25-kDa marker reached the bottom of the gel in order to get a better separation of high-molecular-weight species, and subsequently probed with anti-ISG15 antibody (D). Ponceau staining of the transferred membranes showed equal loading of proteins (data not shown). Conj., conjugate.

FIG. 7.

Detection of ISG15 conjugation to specific target proteins. Plasmid constructs for ISG15 targets (pCAGGS-PLCγ1, pFlagCMV2-serpin 2A, pCEP-HA-Erk1, or pFlagCMV2-AML1B) were transfected to 293T cells in the presence or absence of a protein ISGylation system (6His-mISG15, HA-hUBE1L, and Flag-UbcM8). Cells were harvested 48 h after transfection and analyzed either by direct Western blotting (A) or by reciprocal pull-down assay followed by Western blotting (B, C, D, and E). Plasmids are identified by the ending letters and numbers of each designation. Conj., conjugate; IgG, immunoglobulin G; WB, Western blot; PD, pull-down assay; IP; immunoprecipitation.