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

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.     

The interferon-inducible ubiquitin-protein isopeptide ligase (E3) EFP also functions as an ISG15 E3 ligase

The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3sigma protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3sigma in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3sigma in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations.     

Different roles for two ubiquitin-like domains of ISG15 in protein modification

ISG15 (interferon-stimulated gene 15) is a novel ubiquitin-like (UbL) modifier with two UbL domains in its architecture. We investigated different roles for the two UbL domains in protein modification by ISG15 (ISGylation) and the impact of Influenza B virus NS1 protein (NS1B) on regulation of the pathway. The results show that, although the C-terminal domain is sufficient to link ISG15 to UBE1L and UbcH8, the N-terminal domain is dispensable in the activation and transthiolation steps but required for efficient E3-mediated transfer of ISG15 from UbcH8 to its substrates. NS1B specifically binds to the N-terminal domain of ISG15 but does not affect ISG15 linkage via a thioester bond to its activating and conjugating enzymes. However, it does inhibit the formation of cellular ISG15 conjugates upon interferon treatment. We propose that the N-terminal UbL domain of ISG15 mainly functions in the ligation step and NS1B inhibits ISGylation by competing with E3 ligases for binding to the N-terminal domain.     

泛素样蛋白ISG15抗病毒机制研究进展

ISG15(Interferon stimulated gene 15,ISG15)蛋白是由干扰素诱导产生的一种泛素样蛋白分子,分子量大小约为15kD。ISG15同泛素分子相类似可以被共价结合于其他蛋白分子上,这种现象称为ISG化(ISGylation)现象。ISG化系统包括ISG15、UBE1L、UBCH8和HERC5四类蛋白分子,协同完成ISG化过程。ISG15及ISG化系统在抗病毒反应中具有重要作用。近几年对于ISG15的抗病毒作用和机制的研究已经有了很大的突破,ISG15的抗病毒作用也越来越受到人们重视,了解清楚ISG15抗病毒机制对于研制新的抗病毒药物及提出新的抗病毒策略具有重要意义。本文对ISG15在不同种病毒中的抗病毒机制研究进展进行了简要综述。     

Enhanced antibacterial potential in UBP43-deficient mice against Salmonella typhimurium infection by up-regulating type I IFN signaling

ISG15 is an IFN-inducible ubiquitin-like protein and its expression and conjugation to target proteins are dramatically induced upon viral or bacterial infection. We have generated a UBP43 knockout mouse model that is lacking an ISG15-specific isopeptidase to study the biological role of the protein ISGylation system. We report that UBP43-deficient mice are hypersensitive to LPS-induced lethality and that TIR domain-containing adapter inducing IFN-beta --> IFN regulatory factor 3 --> type I IFN is the major axis to induce protein ISGylation and UBP43 expression in macrophages upon LPS treatment. In ubp43(-/-) macrophages, upon LPS treatment we detected increased expression of IFN-stimulated genes, including genes for several cytokines and chemokines involved in the innate immune response. The ubp43(-/-) mice were able to restrict the growth of Salmonella typhimurium more efficiently than wild-type mice. These results clearly demonstrate two aspects of IFN-signaling, a beneficial effect against pathogens but a detriment to the body without strict control.     

Identification and Herc5-mediated ISGylation of novel target proteins

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.     

类泛素蛋白ISG15及其共价酶修饰系统与肿瘤

干扰素刺激基因15(interferon-stimulated gene 15,ISG15)编码的ISG15蛋白是最早发现的类泛素修饰蛋白。不仅干扰素和病毒感染能诱导该基因的表达,一些抗肿瘤药物也能刺激ISG15的表达。近年来,ISG15及其修饰系统与肿瘤的关系备受关注。研究证实,ISG15的激活酶E1即UBE1L与某些肿瘤的抑制有关,而其他一些成分如ISG15解聚酶UBP43则与肿瘤发生发展有关。此外,ISG15高表达与肿瘤的转移有关,ISG15对化疗药物的敏感性也有影响。本文较为全面地阐述ISG15及其修饰系统在肿瘤抑制或发生发展中的生物学作用,这将增强对ISG15与肿瘤关系的基础性认识,并为发展新的肿瘤靶向性治疗提供理论依据。     

Negative regulation of protein phosphatase 2Cbeta by ISG15 conjugation

ISG15, an interferon-upregulated ubiquitin-like protein, is covalently conjugated to various cellular proteins (ISGylation). In this study, we found that protein phosphatase 2Cbeta (PP2Cbeta), which functions in the nuclear factor kappaB (NF-kappaB) pathway via dephosphorylation of TGF-beta-activated kinase, was ISGylated, and analysis by NF-kappaB luciferase reporter assay revealed that PP2Cbeta activity was suppressed by co-expression of ISG15, UBE1L, and UbcH8. We determined the ISGylation sites of PP2Cbeta and constructed its ISGylation-resistant mutant. In contrast to the wild type, this mutant suppressed the NF-kappaB pathway even in the presence of ISG15, UBE1L, and UbcH8. Thus, we propose that ISGylation negatively regulates PP2Cbeta activity.     

Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.     

HERC6 is the main E3 ligase for global ISG15 conjugation in mouse cells

Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15), thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice. HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its closest relative, HerC6, does not. So far, the requirement of HerC5 for ISG15-mediated antiviral activity has remained unclear. One of the main obstacles to address this issue has been that no HerC5 homologue exists in mice, hampering the generation of a good knock-out model. However, mice do express a homologue of HerC6 that, in contrast to human HerC6, can mediate ISGylation.Here we report that the mouse HerC6 N-terminal RCC1-like domain (RLD) allows ISG15 conjugation when replacing the corresponding domain in the human HerC6 homologue. In addition, sequences in the C-terminal HECT domain of mouse HerC6 also appear to facilitate efficient ISGylation. Mouse HerC6 paralleled human HerC5 in localization and IFN-inducibility. Moreover, HerC6 knock-down in mouse cells abolished global ISGylation, whereas its over expression enhanced the IFNβ promoter and conferred antiviral activity against vesicular stomatitis virus and Newcastle disease virus. Together these data indicate that HerC6 is likely the functional counterpart of human HerC5 in mouse cells, suggesting that HerC6(-/-) mice may provide a feasible model to study the role of human HerC5 in antiviral responses.     

Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection

Innate immune responses provide the host with an early protection barrier against infectious agents, including viruses, and help shape the nature and quality of the subsequent adaptive immune responses of the host. Expression of ISG15 (UCRP), a ubiquitin-like protein, and protein ISGylation are highly increased upon viral infection. We have identified UBP43 (USP18) as an ISG15 deconjugating protease. Protein ISGylation is enhanced in cells deficient in UBP43 (ref. 6). Here we have examined the role of UBP43, encoded by the gene Usp18, in innate immunity to virus infection. Usp18(-/-) mice were resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that developed in wild-type mice after intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV), respectively. Survival of Usp18(-/-) mice after intracerebral LCMV infection correlated with a severe inhibition of LCMV RNA replication and antigen expression in the brain and increased levels of protein ISGylation. Consistent with these findings, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages from Usp18(-/-) mice showed restricted LCMV replication. Moreover, MEF from Usp18(-/-) mice showed enhanced interferon-mediated resistance to the cytopathic effect caused by VSV and Sindbis virus (SNV). This report provides the first direct evidence that the ISG15 protease UBP43 and possibly protein ISGylation have a role in innate immunity against viral infection.     

Microarray analysis reveals that Type I interferon strongly increases the expression of immune-response related genes in Ubp43 (Usp18) deficient macrophages

Type I interferon (IFN) contributes significantly to innate immune responses to pathogen infections in macrophages. Our previous studies demonstrate that Ubp43, an ISG15-specific isopeptidase, is highly expressed in macrophages and noncatalytically inhibits Type I IFN signaling. To understand the effect of Type I IFN and Ubp43 in macrophage activation, we analyzed the expression of IFN-beta stimulated genes in wild-type and Ubp43(-/-) bone marrow derived macrophages (BMMs). Here, we show that Ubp43 regulates IFN-beta stimulated genes at genome level. IFN hypersensitivity of Ubp43(-/-) BMMs resulted in the identification of 749 unique genes that are upregulated by IFN-beta, including a large group of previously unidentified IFN-stimulated genes. Functional analyses of these genes showed that Type I IFN strongly induced the expression of a group of immune response related genes, including genes for antigen presentation, antiviral responses, and chemokine and cytokine production. These results provide excellent biochemical support for the high resistance of viral and bacterial infection of Ubp43 knockout mice, suggesting that Ubp43 is a potential therapeutic target for the enhancement of immune responses against infections.     

ISG15 and immune diseases

ISG15, the product of interferon (IFN)-stimulated gene 15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. ISG15 is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the C-terminal glycine residue before conjugation to target proteins. A set of three-step cascade enzymes, an E1 enzyme (UBE1L), an E2 enzyme (UbcH8), and one of several E3 ligases (e.g., EFP and HERC5), catalyzes ISG15 conjugation (ISGylation) of a specific protein. These enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type I IFNs or other stimuli, such as exposure to viruses and lipopolysaccharide. Mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by ISG15. Some of them are type I IFN-induced proteins, such as PKR and RIG-I, and some are the key regulators that are involved in IFN signaling, such as JAK1 and STAT1, implicating the role of ISG15 and its conjugates in type I IFN-mediated innate immune responses. However, relatively little is known about the functional significance of ISG15 induction due to the lack of information on the consequences of its conjugation to target proteins. Here, we describe the recent progress made in exploring the biological function of ISG15 and its reversible modification of target proteins and thus in their implication in immune diseases.     

The level of hepatitis B virus replication is not affected by protein ISG15 modification but is reduced by inhibition of UBP43 (USP18) expression

Hepatitis B virus (HBV) causes both acute and chronic infection of the human liver and is associated with the development of liver cirrhosis and hepatocellular carcinoma. UBP43 (USP18) is known as an ISG15-deconjugating enzyme and an inhibitor of type I IFN signaling independent of its enzyme activity. In this study, we examined the role of these two previously identified functions of UBP43 in the innate immune response to HBV viral infection. As an in vivo HBV replication model system, a replication-competent DNA construct was injected hydrodynamically into the tail veins of mice. Although the lack of ISG15 conjugation in the absence of ISG15-activating enzyme UBE1L (UBA7) did not affect the level of HBV replication, the steady-state level of HBV DNA was substantially reduced in the UBP43-deficient mice in comparison to the wild-type controls. In addition, introduction of short hairpin RNA against UBP43 resulted in substantially lower levels of HBV DNA at day 4 postinjection and higher levels of ISG mRNAs. These results suggest that HBV infection is more rapidly cleared if UBP43 expression is reduced. Furthermore, these results illustrate the therapeutic potential of modulating UBP43 levels in treating viral infection, especially for viruses sensitive to IFN signaling.     

ISG15 modulates development of the erythroid lineage

Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15(-/-) bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation.     

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

The ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly induced by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. However, how ISGylation contributes to innate immune responses is not clear. The dsRNA-dependent protein kinase (PKR) inhibits translation by phosphorylating eIF2α to exert its anti-viral effect. ISG15 and PKR are induced by interferon, suggesting that a relationship exists between ISGylation and translational regulation. Here, we report that PKR is ISGylated at lysines 69 and 159. ISG15-modified PKR is active in the absence of virus infection and phosphorylates eIF2α to down-regulate protein translation. The present study describes a novel pathway for the activation of PKR and the regulation of protein translation.     

Involvement of UBE1L in ISG15 conjugation during retinoid-induced differentiation of acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) cases expressing the t(15,17) product, promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARalpha), have clinical remissions through leukemic cell differentiation after all-trans-retinoic acid (RA) treatment. This differentiation therapy propelled interest in uncovering molecular mechanisms for RA-dependent APL differentiation. We previously identified the ubiquitin-activating enzyme-E1-like protein (UBE1L) as an RA-regulated target gene in APL that triggers PML/RARalpha degradation and apoptosis. This study reports that conjugation of the ubiquitin-like species, interferon-stimulated gene, 15-kDa protein (ISG15), also occurs during RA-induced APL differentiation. Knock-down of UBE1L expression inhibited this conjugation. RA treatment of APL and other RA-responsive leukemic cells induced expression of UBE1L and ISG15 as well as intracellular ISG15 conjugates. Notably, ISG15 conjugation did not occur in RA-resistant NB4-R1 APL cells. Induction of UBE1L and ISG15 along with ISG15 conjugation in RA-sensitive NB4-S1 APL cells were detected following treatment with specific retinoids and type I interferon (IFN). UBE1L and ISG15 mRNAs were co-expressed in normal human tissues that were examined. In contrast, UBE1L mRNA expression was markedly repressed in several cancer cell lines. A physical association was found between UBE1L and ISG15 in vivo. This required the conserved diglycine motif in the carboxyl terminus of ISG15. Targeting UBE1L expression with small inhibitory RNA or small hairpin RNA inhibited IFN and RA-induced ISG15 conjugation. Formation of ISG15 conjugates through induction of an activating enzyme represents a novel pharmacologic mechanism for regulation of this ubiquitin-related species. Taken together, the observed rela tionship between expression of UBE1L and ISG15, their physical association and coordinate regulation, and induced ISG15 conjugation during leukemic cell differentiation implicate an important role for these proteins in retinoid response.     

Covalent protein modification with ISG15 via a conserved cysteine in the hinge region

The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present.