ISG15 modification of Ubc13 suppresses its ubiquitin-conjugating activity

ISG15 is one of the interferon-stimulated genes and is classified as a ubiquitin-like protein. Upon interferon stimuli, ISG15 is upregulated and becomes conjugated to various cellular proteins (ISGylation). Several target proteins for ISGylation have recently been identified, but the biological consequence of protein ISGylation remains unclear. In the course of our study to identify components of the ISGylation system, we found that Ubc13, an E2 enzyme for ubiquitin conjugation, is covalently modified with ISG15. To determine the meaning of ISGylation of Ubc13, we isolated ISG15-modified Ubc13 protein and compared its ubiquitin-conjugating activity with that of an unmodified one. We found that ISGylation of Ubc13 suppresses its ability to form a thioester intermediate with ubiquitin.     

Negative regulation of ISG15 E3 ligase EFP through its autoISGylation

The function of ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) has been an enigma for many years. Recently, the research of ISGylation has been accelerated by the identification of the enzymes involved in the ISG15 conjugation process. Our previous study identified the interferon inducible protein EFP as an ISG15 isopeptide ligase (E3) for 14-3-3σ. In this study, we show that ISG15 E3 ligase EFP can be modified by ISG15. Two ubiquitin E2 conjugating enzymes, UbcH6 and UbcH8, can support ISGylation of EFP. The Ring-finger domain of EFP is important for its ISGylation. Full-length EFP can enhance the ISGylation of Ring domain deleted EFP, indicating EFP can function as an ISG15 E3 ligase for itself. We also determined the ISGylation site of EFP and created its ISGylation resistant mutant EFP-K117R. Compared to the wild-type EFP, this mutant further increases the ISGylation of 14-3-3σ. Thus we propose that autoISGylation of EFP negatively regulates its ISG15 E3 ligase activity for 14-3-3σ.     

mHERC6 is the essential ISG15 E3 ligase in the murine system

Posttranslational protein modification by ubiquitin and ubiquitin-like modifiers (UBLs) is mediated by a hierarchical cascade of conjugating enzymes and affects multiple biological processes within the cell. Interferon-stimulated gene 15 (ISG15) is an UBL, which is strongly induced by type I Interferon and ISG15 modification was shown to play an essential role in antiviral defense. While hHERC5 is the major E3 ligase for ISG15 modification in humans, ISGylation in the murine systems at the level of E3 ligases was weakly characterized as rodent genomes lack a direct homologue of hHERC5. Here, we show that mHERC6 is strongly induced by different pathogen-associated molecular patterns (PAMPs) in a type I Interferon receptor (IFNAR1) dependent manner. We demonstrate that mHERC6 is essential for endogenous murine ISGylation and thus represents the dominant ISG15 E3 ligase in mice. In contrast to its human homologue, mHERC6 is also capable to mediate conjugation of human ISG15.     

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.     

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.     

Nitrosylation of ISG15 prevents the disulfide bond-mediated dimerization of ISG15 and contributes to effective ISGylation

The expression of the ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly activated by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. Inducible nitric-oxide synthase (iNOS) is induced by the similar stimuli as ISG15 and enhances the production of nitric oxide (NO), a pleiotropic free radical with antipathogen activity. Here, we report that cysteine residues (Cys-76 and -143 in mouse, Cys-78 in human) of ISG15 can be modified by NO, and the NO modification of ISG15 decreases the dimerization of ISG15. The mutation of the cysteine residue of ISG15 to serine improves total ISGylation. The NO synthase inhibitor S-ethylisothiourea reduces endogenous ISGylation. Furthermore, ectopic expression of iNOS enhanced total ISGylation. Together, these results suggest that nitrosylation of ISG15 enhances target protein ISGylation. This is the first report of a relationship between ISGylation and nitrosylation.     

Proteomic identification of proteins conjugated to ISG15 in mouse and human cells

Though the interferon-inducible protein ISG15 was one of the first ubiquitin-like modifiers to be discovered, much remains unknown about the identity of proteins conjugated to ISG15 or the biologic consequences of modification. To gain a better understanding of the cellular pathways affected by ISG15, we identified proteins targeted for ISGylation using a proteomic approach. Mass spectrometric analysis identified 76 candidate ISGylation targets in anti-ISG15 immunoprecipitates from interferon-treated mouse or human cells. Twenty-one proteins were found in both mouse and human samples, including STAT1, a known target of ISGylation. Candidates identified in both species were tested for ISGylation in a transfection system: 18 of 19 proteins tested were ISGylated in this system. Two candidates, EF-2 and VCP, were also shown to be ISGylated in an interferon-dependent manner in the absence of exogenous over-expression. Seven proteins identified from a single species, but functionally related to candidates found in both species, were also ISGylated in the over-expression system. Proteins that can be ISGylated play important roles in translation, glycolysis, stress responses, and cell motility. These data indicate that ISGylation targets proteins found in several fundamentally important cellular pathways and will contribute to understanding the physiologic role of interferon-induced ISG15 and ISG15 conjugation.     

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.     

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.     

Ube1L and protein ISGylation are not essential for alpha/beta interferon signaling

The expression of ubiquitin-like modifier ISG15 and its conjugation to target proteins are highly induced by interferon (IFN) stimulation and during viral and bacterial infections. However, the biological significance of this modification has not been clearly understood. To investigate the function of protein modification by ISG15, we generated a mouse model deficient in UBE1L, an ISG15-activating enzyme. Ube1L-/- mice did not produce ISG15 conjugates but expressed free ISG15 normally. ISGylation has been implicated in the reproduction and innate immunity. However, Ube1L-/- mice were fertile and exhibited normal antiviral responses against vesicular stomatitis virus and lymphocytic choriomeningitis virus infection. Our results indicate that UBE1L and protein ISGylation are not critical for IFN-alpha/beta signaling via JAK/STAT activation. Moreover, using Ube1L/Ubp43 double-deficient mice, we showed that lack of UBP43, but not the increase of protein ISGylation, is related to the increased IFN signaling in Ubp43-deficient mice.     

Proteasomes modulate conjugation to the ubiquitin-like protein,ISG15

ISG15 is a ubiquitin-like protein that is induced by interferon and microbial challenge. Ubiquitin-like proteins are covalently conjugated to cellular proteins and may intersect the ubiquitin-proteasome system via common substrates or reciprocal regulation. To investigate the relationship between ISG15 conjugation and proteasome function, we treated interferon-induced cells with proteasome inhibitors. Surprisingly, inhibition of proteasomal, but not lysosomal, proteases dramatically enhanced the level of ISG15 conjugates. The stimulation of ISG15 conjugates occurred rapidly in the absence of protein synthesis and was most dramatic in the cytoskeletal protein fraction. Inhibition of ISG15 conjugation by ATP depletion abrogated the proteasome inhibitor-dependent increase in ISG15 conjugates, suggesting that the effect was mediated by de novo conjugation, rather than protection from proteasomal degradation or inhibition of ISG15 deconjugating activity. The increase in ISG15 conjugates did not occur through a stabilization of the ISG15 E1 enzyme, UBE1L. Furthermore, simultaneous modification of proteins by both ISG15 and ubiquitin did not account for the proteasome inhibitor-dependent increase in ISG15 conjugates. These findings provide the first evidence for a link between ISG15 conjugation and proteasome function and support a model in which proteins destined for ISG15 conjugation are proteasome-regulated.     

Modification of BECN1 by ISG15 plays a crucial role in autophagy regulation by type I IFN/interferon

ISG15 (ISG15 ubiquitin-like modifier), a ubiquitin-like protein, is one of the major type I IFN (interferon) effector systems. ISG15 can be conjugated to target proteins (ISGylation) via the stepwise action of E1, E2, and E3 enzymes. Conjugated ISG15 can be removed (deISGylated) from target proteins by USP18 (ubiquitin-specific peptidase 18). Here we investigated the role of deISGylation by USP18 in regulating autophagy and EGFR degradation in cells treated with type I IFNs. We show that type I IFN induced expression of ISG15 leads to ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 which competes with Lys63 ubiquitination of BECN1. We demonstrate that ISGylation of BECN1 at Lys117, as well as Lys263, Lys265, and Lys266 serve an important role in negative regulation of intracellular processes including autophagy and EGFR degradation that are critically dependent upon the activity of class III PtdIns 3-kinase. Our studies provide fundamental new mechanistic insights into the innate immunity response implemented by type I IFNs.     

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.     

The ISG15/USP18 ubiquitin-like pathway (ISGylation system) in hepatitis C virus infection and resistance to interferon therapy

The ISG15/USP18 pathway modulates cellular functions and is important for the host innate immune response to chronic viral infections such as Hepatitis C Virus (HCV). Interferon stimulated gene 15 (ISG15) was the first ubiquitin-like protein modifier identified. As in ubiquitination, ISG15 conjugates to target proteins (ISGylation) through the sequential enzymatic action of activating E1, conjugating E2, and ligating E3 enzymes. ISGylation modulates signal transduction pathways and host anti-viral response. The ISGylation process is reversible through the action of an ISG15 protease, USP18. Ubiquitin-like specific protease 18 (USP18) has functions that are both ISG15-dependent and ISG15-independent; the importance of the ISG15/USP18 pathway to chronic HCV infection is illustrated by the consistent finding of increased levels of ISG15 and USP18 in the liver tissue of patients who do not respond to interferon-based treatments. Mechanistically, HCV seems to exploit the ISG15/USP18 pathway to promote viral replication and evade innate anti-viral immune responses.     

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.     

Interferon-stimulated gene 15 enters posttranslational modifications of p53

The tumor suppressor protein p53 is a central governor of various cellular signals. It is well accepted that ubiquitination as well as ubiquitin-like (UBL) modifications of p53 protein is critical in the control of its activity. Interferon-stimulated gene 15 (ISG15) is a well-known UBL protein with pleiotropic functions, serving both as a free intracellular molecule and as a modifier by conjugating to target proteins. Initially, attentions have historically focused on the antiviral effects of ISG15 pathway. Remarkably, a significant role in the processes of autophagy, DNA repair, and protein translation provided considerable insight into the new functions of ISG15 pathway. Despite the deterministic revelation of the relation between ISG15 and p53, the functional consequence of p53 ISGylation appears somewhat confused. More important, more recent studies have hinted p53 ubiquitination or other UBL modifications that might interconnect with its ISGylation. Here, we aim to summarize the current knowledge of p53 ISGylation and the differences in other significant modifications, which would be beneficial for the development of p53-based cancer therapy.     

Cell type-dependent regulation of free ISG15 levels and ISGylation

Interferon-stimulated gene 15 (ISG15) is an ubiquitin-like protein, which can either be found as a free protein or covalently-bound to target proteins via ISGylation. The functions of free and conjugated ISG15 are ambiguous in tumorigenesis owing to its roles as an oncogene and a tumour suppressor gene. This dual role for ISG15 could be a result of the cancer cell type and the cellular context. Here, we report that ISG15 expression is upregulated in different cancer cells compared to normal cells. Furthermore, we found higher endogenous, free ISG15 protein levels in MCF7 breast cancer cells than in other cells, suggesting that non-conjugated ISG15 levels are cell type-specific. Additionally, we demonstrated that interferon gamma (IFN-?) increased both free and conjugated levels of ISG15 in MCF7 cells. Interestingly, endogenous conjugated and free ISG15 levels were differentially regulated by IFN-? in several cell lines. On characterisation of the subcellular distribution of ISG15 in several cell types, our results indicated that free ISG15 was mainly localised to the cytoplasm of MCF7 cells, whereas ISGylation marks were also found in the cytoplasm, but mainly in the nucleus, with a specific distribution pattern in each cell type. Thus, free and conjugated ISG15 protein levels and their subcellular distribution are cell type-dependent, whereas IFN-? signalling may differentially control the abundance of both ISG15 forms in transformed and normal cells.     

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。     

The hinge region between two ubiquitin-like domains destabilizes recombinant ISG15 in solution

Interferon-stimulated gene (ISG) 15 mediates antiviral responses and also is upregulated within the endometrium in response to the developing embryo during early pregnancy. Structurally, ISG15 resembles two ubiquitin domains (30% identical) that are separated by a hinge region. Recombinant (r) bovISG15 is not stable in solution. It was hypothesized that the hinge region contributed to the instability of rbovISG15. Within 24 h of dialysis, rbovISG15 formed complexes as detected by reducing and denaturing SDS-PAGE. However, chemical perturbations of cysteine prevented formation of rbovISG15 complexes over time. Furthermore, a site-directed mutant of rbovISG15 (Cys80Ser) was isomeric and more stable than rbovISG15. Neither wild-type nor mutant rbovISG15 was able to interact with the ISG15 E1 initiating enzyme, UBE1L, in an in vitro pull-down assay. Ovine (ov) ISG15 has three additional amino acids within the hinge region that were hypothesized to increase stability and the degree of interaction with UBE1L because of increased separation of the ubiquitin-like domains. Over time in solution, rovISG15 the level of rovISG15 secondary structure was diminished, whereas the Cys80Ser rovISG15 structure did not change. A GST-Cys80Ser rovISG15 fusion protein had increased structural stability and enhanced protein-protein interaction with UBE1L after dialysis for 48 h, when compared to the GST-rovISG15 fusion protein or rbovISG15. Models of bovISG15, Cys80Ser bovISG15, and ovISG15 were constructed, which confirmed that the hinge region between the two ubiquitin domains destabilizes rbovISG15 in solution.     

Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.