The family of ubiquitin-like molecules (UbLs) comprises several members, each of which has sequence, structural, or functional similarity to ubiquitin. ISG15 is a homolog of ubiquitin in vertebrates and is strongly upregulated following induction by type I interferon. ISG15 can be covalently attached to proteins, analogous to ubiquitination and with actual support of ubiquitin conjugating factors. Specific proteases are able to reverse modification with ubiquitin or UbLs by hydrolyzing the covalent bond between their C-termini and substrate proteins. The tail regions of ubiquitin and ISG15 are identical and we therefore hypothesized that promiscuous deubiquitinating proteases (DUBs) might exist, capable of recognizing both ubiquitin and ISG15.
We have cloned and expressed 22 human DUBs, representing the major clades of the USP protease family. Utilizing suicide inhibitors based on ubiquitin and ISG15, we have identified USP2, USP5 (IsoT1), USP13 (IsoT3), and USP14 as ISG15-reactive proteases, in addition to the bona fide ISG15-specific protease USP18 (UBP43). USP14 is a proteasome-associated DUB, and its ISG15 isopeptidase activity increases when complexed with the proteasome.
By evolutionary standards, ISG15 is a newcomer among the UbLs and it apparently not only utilizes the conjugating but also the deconjugating machinery of its more established relative ubiquitin. Functional overlap between these two posttranslational modifiers might therefore be more extensive than previously appreciated and explain the rather innocuous phenotype of ISG15 null mice.
Coronaviruses express a deubiquitinating protein, the papain-like protease-2 (PLP2), that removes both ubiquitin and the ubiquitin-like interferon (IFN)-stimulated gene 15 (ISG15) protein from target proteins. ISG15 has antiviral activity against a number of viruses; therefore, we examined the effect of ISG15 conjugation (ISGylation) in a model of acute viral hepatitis induced by the murine hepatitis virus strain 3 (MHV-3) coronavirus. Mice deficient in the ISG15 deconjugating enzyme, ubiquitin-specific peptidase-18 (USP18), accumulate high levels of ISG15-conjugated proteins and are hypersensitive to type I IFN. Infecting USP18−/− mice with MHV-3 resulted in extended survival (8 ± 1.2 versus 4 days) and in improved liver histology, a decreased inflammatory response, and viral titers 1 to 2 logs lower than in USP18+/+ mice. The suppression of viral replication was not due to increased IFN since infected USP18−/− mice had neither increased hepatic IFN-α, -β, or -γ mRNA nor circulating protein. Instead, delayed MHV-3 replication coincided with high levels of cellular ISGylation. Decreasing ISGylation by knockdown of the ISG15 E1 enzyme, Ube1L, in primary USP18+/+ and USP18−/− hepatocytes led to increased MHV-3 replication. Both in vitro and in vivo, increasing MHV-3 titers were coincident with increased PLP2 mRNA and decreased ISGylation over the course of infection. The pharmacologic inhibition of the PLP2 enzyme in vitro led to decreased MHV-3 replication. Overall, these results demonstrate the antiviral effect of ISGylation in an in vivo model of coronavirus-induced mouse hepatitis and illustrate that PLP2 manipulates the host innate immune response through the ISG15/USP18 pathway.
IMPORTANCE There have been a number of serious worldwide pandemics due to widespread infections by coronavirus. This virus (in its many forms) is difficult to treat, in part because it is very good at finding “holes” in the way that the host (the infected individual) tries to control and eliminate the virus. In this study, we demonstrate that an important host viral defense—the ISG15 pathway—is only partially effective in controlling severe coronavirus infection. Activation of the pathway is very good at suppressing viral production, but over time the virus overwhelms the host response and the effects of the ISG15 pathway. These data provide insight into host-virus interactions during coronavirus infection and suggest that the ISG15 pathway is a reasonable target for controlling severe coronavirus infection although the best treatment will likely involve multiple pathways and targets.
ISG15 is an ubiquitin-like protein that is induced rapidly by interferon stimulation. Like ubiquitin, ISG15 forms covalent conjugates with its target proteins in a process called ISGylation, which in mammals is known to play a role in antiviral immunity. In contrast to mammalian ISG15, the function of teleost ISG15 is unclear. In this study, we identified and analyzed the function of an ISG15 homologue, CsISG15, from tongue sole (Cynoglossus semilaevis). CsISG15 is composed of 162 residues and possesses two tandem ubiquitin-like domains and the highly conserved LRGG motif found in all known ISG15. Expression of CsISG15 occurred in a wide range of tissues and was upregulated in kidney and spleen by viral and bacterial infection. In vitro study with primary head kidney (HK) lymphocytes showed that megalocytivirus infection caused induction of CsISG15 expression and extracellular release of CsISG15 protein. Purified recombinant CsISG15 (rCsISG15) activated HK macrophages and enhanced the expression of immune genes in HK lymphocytes, both these effects, however, were significantly reduced when the conserved LRGG sequence was mutated to LAAG. Further study showed that the presence of rCsISG15 during megalocytivirus infection of HK lymphocytes reduced intracellular viral load, whereas antibody blocking of CsISG15 enhanced viral infection. Likewise, interference with CsISG15 expression by RNAi promoted viral infection. Taken together, these results indicate that CsISG15, a teleost ISG15, promotes antiviral immune response and that, unlike mammalian ISG15, CsISG15 exerts its immunoregulatory effect in the form of an unconjugated extracellular cytokine. In addition, these results also suggest a role for the LRGG motif other than that in protein conjugation.
Isg15 covalently modifies murine endometrial proteins in response to early pregnancy. Isg15 can also be severed from targeted proteins by a specific protease called Ubp43 (Usp18). Mice lacking Ubp43 (null) form increased conjugated Isg15 in response to interferon. The Isg15 system has not been examined in chorioallantoic placenta (CP) or mesometrial (MM) components of implantation sites beyond 9.5 days post coitum (dpc). It was hypothesized that deletion of Ubp43 would cause disregulation of Isg15 in implantation sites, and that this would affect pregnancy rates.
Heterozygous (het) Ubp43 mice were mated and MM and CP implantation sites were collected on 12.5 and 17.5 days post-coitum (dpc).
Free and conjugated Isg15 were greater on 12.5 versus 17.5 dpc in MM. Free and conjugated Isg15 were also present in CP, but did not differ due to genotype on 12.5 dpc. However, null CP had greater free and conjugated Isg15 when compared to het/wt on 17.5 dpc. Null progeny died in utero with fetal genotype ratios (wt:het:null) of 2:5:1 on 12.5 and 2:2:1 on 17.5 dpc. Implantation sites were disrupted within the junctional zone and spongiotrophoblast, contained less vasculature based on lectin B4 staining and contained greater Isg15 mRNA and VEGF protein in Ubp43 null when compared to wt placenta.
It is concluded that Isg15 and its conjugates are present in implantation sites during mid to late gestation and that deletion of Ubp43 causes an increase in free and conjugated Isg15 at the feto-maternal interface. Also, under mixed genetic background, deletion of Ubp43 results in fetal death.
ISG15 is an interferon-induced ubiquitin-like modifier which can be conjugated to distinct, but largely unknown, proteins. ISG15 has been implicated in a variety of biological activities, which encompass antiviral defense, immune responses, and pregnancy. Mice lacking UBP43 (USP18), the ISG15-deconjugating enzyme, develop a severe phenotype with brain injuries and lethal hypersensitivity to poly(I:C). It has been reported that an augmented conjugation of ISG15 in the absence of UBP43 induces prolonged STAT1 phosphorylation and that the ISG15 conjugation plays an important role in the regulation of JAK/STAT and interferon signaling (O. A. Malakhova, M. Yan, M. P. Malakhov, Y. Yuan, K. J. Ritchie, K. I. Kim, L. F. Peterson, K. Shuai, and D. E. Zhang, Genes Dev. 17:455-460, 2003). Here, we report that ISG15−/− mice are viable and fertile and display no obvious abnormalities. Lack of ISG15 did not affect the development and composition of the main cellular compartments of the immune system. The interferon-induced antiviral state and immune responses directed against vesicular stomatitis virus and lymphocytic choriomeningitis virus were not significantly altered in the absence of ISG15. Furthermore, interferon- or endotoxin-induced STAT1 tyrosine-phosphorylation, as well as expression of typical STAT1 target genes, remained unaffected by the lack of ISG15. Thus, ISG15 is dispensable for STAT1 and interferon signaling.
Dendritic cells (DCs) represent the key cells linking innate and adaptive immune responses. It is critical to understand the molecular factors regulating DC differentiation. Usp18 is an interferon (IFN)-inducible member of the ubiquitin-specific protease (USP) family, which deconjugates ubiquitin-like modifier ISG15 from target proteins, and competitively inhibits IFN-α/β-induced JAK/STAT activation. This studydemonstrates that the frequency of conventional CD11b+ DCs in the spleen of Usp18−/− mice was significantly reduced, while the frequencies of conventional CD8+ DCs and plasmacytoid DCs remained normal. In addition, Usp18−/− bone marrow (BM) cells generate DCs less efficiently in GM-CSF-supplemented culture, demonstrating a fundamental defect throughout the DC differentiation pathway. Usp18−/− BM cells were rescued by exogenous expression of either wild type, or deconjugation-inactive, Usp18, while superimposition of an IFN-α/β receptor knockout returned in vivo DC populations to normal, clearly showing that the defect seen is due solely to Usp18’s effect on IFN signaling. Finally, Usp18−/− BM-DCs expressed high levels of SOCS1/SOCS3, known inhibitors of GM-CSF signaling, providing a mechanistic explanation for the phenotype. In conclusion, we have identified a novel role of Usp18 in modulating conventional CD11b+ DC development via its inhibitory effect on Type I interferon signaling.
Usp18; Ubp43; interferon; conventional dendritic cells; SOCS proteins; GM-CSF; pSTAT5
UBP43/USP18 was described as a specific protease that removes conjugated ubiquitin-like modifier ISG15 from target proteins. The severe phenotype of UBP43−/− mice characterized by premature death, brain cell injury, and deregulated STAT1 signaling was ascribed to an enhanced conjugation of ISG15. In contrast, no phenotypic changes were detected in ISG15−/− mice. To verify the role of ISG15 in the phenotype of UBP43−/− mice, we employed mice deficient for both ISG15 and UBP43. Here, we show that the phenotype of UBP43−/− mice was not rescued by the absence of ISG15, as evident from unchanged mortality, neurological symptoms, and occurrence of hydrocephalus. Also, the reported hypersensitivity of UBP43−/− mice to an interferon inducer, poly(I · C), was ISG15 independent. Furthermore, no evidence for a role of ISG15 in the modulation of STAT1 signaling or in the resistance against lymphocytic choriomeningitis virus and vesicular stomatitis virus was found. Presented results clearly demonstrate that the phenotypic alterations of UBP43−/− mice are not caused by the lack of ISG15 deconjugation and must be due to another, non-ISG15-mediated molecular mechanism.
Modification of proteins by ubiquitin (Ub)-like proteins (UBLs) plays an important role in many cellular processes, including cell cycle progression, nuclear transport, and autophagy. Protein modification occurs via UBL-conjugating and -deconjugating enzymes, which presumably exert a regulatory function by determining the conjugation status of the substrate proteins. To target and identify UBL-modifying enzymes, we produced Nedd8, ISG15, and SUMO-1 in Escherichia coli and equipped them with a C-terminal electrophilic trap (vinyl sulfone [VS]) via an intein-based method. These C-terminally modified UBL probes reacted with purified UBL-activating (E1), -conjugating (E2), and -deconjugating enzymes in a covalent fashion. Modified UBLs were radioiodinated and incubated with cell lysates prepared from mouse cell lines and tissues to allow visualization of polypeptides reactive with individual UBL probes. The cell type- and tissue-specific labeling patterns observed for the UBL probes reflect distinct expression profiles of active enzymes, indicating tissue-specific functions of UBLs. We identify Ub C-terminal hydrolase L1 (UCH-L1) and DEN1/NEDP1/SENP8, in addition to UCH-L3, as proteases with specificity for Nedd8. The Ub-specific protease isopeptidase T/USP5 is shown to react with ISG15-VS. Furthermore, we demonstrate that the desumoylation enzyme SuPr-1 can be modified by SUMO-1-VS, a modification that is dependent on the SuPr-1 active-site cysteine. The UBL probes described here will be valuable tools for the further characterization of the enzymatic pathways that govern modification by UBLs.
Acute promyelocytic leukemia (APL) is characterized by expression of promyelocytic leukemia (PML)/retinoic acid (RA) receptor α (RARα) protein and all-trans-RA-mediated clinical remissions. RA treatment can confer PML/RARα degradation, overcoming dominant-negative effects of this oncogenic protein. The present study uncovered independent retinoid degradation mechanisms, targeting different domains of PML/RARα. RA treatment is known to repress PML/RARα and augment ubiquitin-activating enzyme-E1-like (UBE1L) protein expression in NB4-S1 APL cells. We previously reported RA-induced UBE1L and the IFN-stimulated gene, 15-kDa protein ISG15ylation in APL cells. Whether the ubiquitin-like protein ISG15 directly conjugates with PML/RARα was not explored previously and is examined in this study. Transient transfection experiments with different PML/RARα domains revealed that RA treatment preferentially down-regulated the RARα domain, whereas UBE1L targeted the PML domain for repression. As expected, ubiquitin-specific protease 18 (UBP43/USP18), the ISG15 deconjugase, opposed UBE1L but not RA-dependent PML/RARα degradation. In contrast, the proteasomal inhibitor, N-acetyl-leucinyl-leucinylnorleucinal, inhibited both UBE1L- and RA-mediated PML/RARα degradation. Notably, UBE1L induced ISG15ylation of the PML domain of PML/RARα, causing its repression. These findings confirmed that RA triggers PML/RARα degradation through different domains and distinct mechanisms. Taken together, these findings advance prior work by establishing two pathways converge on the same oncogenic protein to cause its degradation and thereby promote antineoplastic effects. The molecular pharmacologic implications of these findings are discussed.
Adenoviral replication depends on viral as well as cellular proteins. However, little is known about cellular proteins promoting adenoviral replication. In our screens to identify such proteins, we discovered a cellular component of the ubiquitin proteasome pathway interacting with the central regulator of adenoviral replication. Our binding assays mapped a specific interaction between the N-terminal domains of both viral E1B-55K and USP7, a deubiquitinating enzyme. RNA interference-mediated downregulation of USP7 severely reduced E1B-55K protein levels, but more importantly negatively affected adenoviral replication. We also succeeded in resynthesizing an inhibitor of USP7, which like the knockdown background reduced adenoviral replication. Further assays revealed that not only adenoviral growth, but also adenoviral oncogene-driven cellular transformation relies on the functions of USP7. Our data provide insights into an intricate mechanistic pathway usurped by an adenovirus to promote its replication and oncogenic functions, and at the same time open up possibilities for new antiviral strategies.
Adenoviral infections can result in severe outcomes leading to mortality especially in children undergoing immunosuppressive therapies. Unfortunately, no specific anti-adenoviral treatments are available to treat disseminated adenoviral infections. We have set out to identify host factors promoting adenoviral growth and could identify the cellular protein Ubiquitin-specific protease 7 (USP7) being central to adenoviral infection. Here we show that USP7 interacts with the viral protein E1B-55K, a central regulator of adenoviral replication and adenoviral oncogene-mediated cellular transformation. We demonstrate that USP7 ensures stability and/or proper expression levels of adenoviral proteins at early and late time points of infection. Consistent with this, small-molecule inhibitors of USP7 showed efficient reduction of capsid protein levels and viral progeny numbers. Thus, USP7 inhibition might be a useful treatment option in the context of disseminated adenoviral infections. Moreover, we were also able to show that adenoviral oncogene-mediated cellular transformation can be hampered by USP7 disruption. In summary, this study shows that two different adenoviral disease mechanisms can be inhibited by targeting one host cellular factor.
The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVΔE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15−/− cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVΔE3L caused significant disease and mortality, an effect not observed in VVΔE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVΔE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor.
Modification of proteins by ubiquitin (UB) and ubiquitin-like proteins (UBL) represents a key regulatory process of innate and adaptive immune responses. Interferon-stimulated gene product 15 (ISG15) is a member of UBL molecules that can reversibly be conjugated to proteins mediating considerable antiviral response. In turn, many viruses, including poxviruses, have evolved strategies to block the antiviral and inflammatory effects of innate immune responses to keep cells alive until virus replication is completed. Here, a novel viral immune evasion mechanism that inhibits ISG15-dependent antiviral pathway is described. Vaccinia virus (VACV) pathogenesis in ISG15+/+ versus ISG15−/− mice is linked to the virus E3 protein, blocking the activity of ISG15 through its C-terminal domain. This effect was independent of PKR activation. ISG15 controls the inflammatory response regulating cytokine levels. Our findings support a new strategy for poxviruses to evade the host antiviral response through interaction of the virus E3 protein with ISG15.
ISG15, a 15-kDa interferon-induced protein that participates in antiviral defenses of mammals, is highly conserved among vertebrates. In fish, as in mammals, viral infection and interferon treatment induce isg15 expression. The two ubiquitin-like domains of ISG15 and the presence of a consensus LRLRGG sequence in the C-terminal region, which is required for the covalent conjugation to a substrate protein, are also conserved in fish. Our data demonstrate that overexpression of zebrafish ISG15 (zf-ISG15) in EPC cells is sufficient to inhibit viral infection by RNA viruses belonging to the genera Novirhabdovirus and Birnavirus and by DNA viruses of the genus Iridovirus. In coexpression experiments with IHNV proteins, we demonstrate specific ISGylation of phosphoprotein and nonvirion protein. Mutation of the glycine residues in the consensus LRLRGG motif abolishes zf-ISG15 conjugation to these proteins and the cellular protection against viral infection, thus connecting ISGylation and ISG15-dependent viral restriction. Additionally, zf-ISG15 overexpression triggers induction of the rig-I and viperin genes as well as, to a lesser extent, the IFN gene. Overall, our data demonstrate the antiviral effect of a fish ISG15 protein, revealing the conservation among vertebrates of an ISGylation mechanism likely directed against viruses. Furthermore, our findings indicate that zf-ISG15 affects the IFN system at several levels, and its study shall shed further light on the evolution of the complex regulation of the innate antiviral response in vertebrate cells.
Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a papain-like protease (PLpro) with both deubiquitinating (DUB) and deISGylating activities that are proposed to counteract the post-translational modification of signaling molecules that activate the innate immune response. Here we examine the structural basis for PLpro's ubiquitin chain and interferon stimulated gene 15 (ISG15) specificity. We present the X-ray crystal structure of PLpro in complex with ubiquitin-aldehyde and model the interaction of PLpro with other ubiquitin-chain and ISG15 substrates. We show that PLpro greatly prefers K48- to K63-linked ubiquitin chains, and ISG15-based substrates to those that are mono-ubiquitinated. We propose that PLpro's higher affinity for K48-linked ubiquitin chains and ISG15 stems from a bivalent mechanism of binding, where two ubiquitin-like domains prefer to bind in the palm domain of PLpro with the most distal ubiquitin domain interacting with a “ridge” region of the thumb domain. Mutagenesis of residues within this ridge region revealed that these mutants retain viral protease activity and the ability to catalyze hydrolysis of mono-ubiquitin. However, a select number of these mutants have a significantly reduced ability to hydrolyze the substrate ISG15-AMC, or be inhibited by K48-linked diubuiquitin. For these latter residues, we found that PLpro antagonism of the nuclear factor kappa-light-chain-enhancer of activated B-cells (NFκB) signaling pathway is abrogated. This identification of key and unique sites in PLpro required for recognition and processing of diubiquitin and ISG15 versus mono-ubiquitin and protease activity provides new insight into ubiquitin-chain and ISG15 recognition and highlights a role for PLpro DUB and deISGylase activity in antagonism of the innate immune response.
All coronaviruses such as the SARS virus and the recently identified Middle East Respiratory Syndrome (MERS) virus encode in their genomes at least one papain-like protease (PLpro) enzyme that has two distinct functions in viral pathogenesis. The first function is to process the viral polyprotein into individual proteins that are essential for viral replication. The second function is to remove ubiquitin and ISG15 proteins from host cell proteins, which likely helps coronaviruses short circuit the host's innate immune response. The 3-dimensional structure of SARS virus PLpro in complex with a human ubiquitin analog was determined and reveals how coronavirus PLpro enzymes strip ubiquitin and ISG15 from host cell proteins at the molecular level. A series of amino acid residues involved in interactions between PLpro and ubiquitin were mutated to identify which interactions are important only for the recognition of ubiquitin and ISG15 modified proteins by PLpro and not for recognition and cleaving of the viral polyprotein. The 3D structure of SARS PLpro with ubiquitin-aldehyde sheds significant new light into how PLpro interacts with ubiquitin-like molecules and provides a molecular road map for performing similar studies on other deadly coronaviruses such as MERS.
The interferon (IFN)-stimulated genes (ISGs) ISG-49, ISG-54, and ISG-56 are highly responsive to viral infection, yet the regulation and function of these genes in vivo are unknown. We examined the simultaneous regulation of these ISGs in the brains of mice during infection with either lymphocytic choriomeningitis virus (LCMV) or West Nile virus (WNV). Expression of the ISG-49 and ISG-56 genes increased significantly during LCMV infection, being widespread and localized predominantly to common as well as distinct neuronal populations. Expression of the ISG-54 gene also increased but to lower levels and with a more restricted distribution. Although expression of the ISG-49, ISG-54, and ISG-56 genes was increased in the brains of LCMV-infected STAT1 and STAT2 knockout (KO) mice, this was blunted, delayed, and restricted to the choroid plexus, meninges, and endothelium. ISG-56 protein was regulated in parallel with the corresponding RNA transcript in the brain during LCMV infection in wild-type and STAT KO mice. Similar changes in ISG-49, ISG-54, and ISG-56 RNA levels and ISG-56 protein levels were observed in the brains of wild-type mice following infection with WNV. Thus, the ISG-49, ISG-54, and ISG-56 genes are coordinately upregulated in the brain during LCMV and WNV infection; this upregulation, in the case of LCMV, was totally (neurons) or partially (non-neurons) dependent on the IFN-signaling molecules STAT1 and STAT2. These findings suggest a dominant role for the ISG-49, ISG-54, and ISG-56 genes in the host response to different viruses in the central nervous system, where, particularly in neurons, these genes may have nonredundant functions.
Intracellular ISG15 is an interferon (IFN)-α/β-inducible ubiquitin-like modifier which can covalently bind other proteins in a process called ISGylation; it is an effector of IFN-α/β-dependent antiviral immunity in mice1–4. We previously published a study describing humans with inherited ISG15 deficiency but without unusually severe viral diseases5. We showed that these patients were prone to mycobacterial disease and that human ISG15 was non-redundant as an extracellular IFN-γ-inducing molecule. We show here that ISG15-deficient patients also display unanticipated cellular, immunological and clinical signs of enhanced IFN-α/β immunity, reminiscent of the Mendelian autoinflammatory interferonopathies Aicardi–Goutières syndrome and spondyloenchondrodysplasia6–9.We further show that an absence of intracellular ISG15 in the patients’ cells prevents the accumulation of USP1810,11, a potent negative regulator of IFN-α/β signalling, resulting in the enhancement and amplification of IFN-α/β responses. Human ISG15, therefore, is not only redundant for antiviral immunity, but is a key negative regulator of IFN-α/β immunity. In humans, intracellular ISG15 is IFN-α/β-inducible not to serve as a substrate for ISGylation-dependent antiviral immunity, but to ensure USP18-dependent regulation of IFN-α/β and prevention of IFN-α/β-dependent autoinflammation.
Host genetics plays a key role in susceptibility to Salmonella Typhimurium infection. We previously used N-ethyl-N-nitrosourea (ENU) mutagenesis to identify a loss of function mutation within the gene ubiquitin specific peptidase 18 (Usp18Ity9), which confers increased susceptibility to Salmonella Typhimurium. USP18 functions to regulate type I IFN signaling and as a protease to remove ISG15 from substrate proteins. Usp18Ity9 mice are susceptible to infection with Salmonella Typhimurium and have increased expression and function of ISG15, but Usp18Ity9 mice lacking Isg15 do not show improved survival with Salmonella challenge. Type I IFN signaling is increased in Usp18Ity9 mice and inhibition of type I IFN signaling is associated with improved survival in mutant mice. Hyperactivation of type I IFN signaling leads to increased IL-10, deregulated expression of autophagy markers, and elevated IL-1β and IL-17. Furthermore, Usp18Ity9 mice are more susceptible to infection with Mycobacterium tuberculosis, have increased bacterial load in lung and spleen, elevated inflammatory cytokines and more severe lung pathology. These findings demonstrate that regulation of type I IFN signaling is the predominant mechanism affecting the susceptibility of Usp18Ity9 mice to Salmonella infection and that hyperactivation of signaling leads to increased IL-10, deregulation of autophagic markers and increased proinflammatory cytokine production.
PMID: 24807690 CAMSID: cams4329
USP18; innate immunity; Salmonella; mycobacteria; type I IFN; ISGylation; autophagy
Type I interferon (alpha/beta interferon [IFN-α/β]) stimulates the expression of interferon-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein, ISG15. Free ISG15 and ISG15 conjugates function in diverse cellular pathways, particularly regulation of antiviral innate immune responses. In this study, we demonstrate that ISG15 overexpression inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication in cell culture and that the antiviral activity of interferon is reduced by inhibition of ISG15 conjugation. PRRSV nonstructural protein 2 (nsp2) was previously identified as a potential antagonist of ISG15 production and conjugation. The protein contains a papain-like protease domain (PLP2) that plays a crucial role in the proteolytic cleavage of the PRRSV replicase polyproteins. PLP2 was also proposed to belong to the ovarian tumor domain-containing superfamily of deubiquitinating enzymes (DUBs), which is capable of inhibiting ISG15 production and counteracting ISG15 conjugation to cellular proteins. To determine whether this immune antagonist function could be selectively inactivated, we engineered a panel of mutants with deletions and/or mutations at the N-terminal border of the nsp2 PLP2-DUB domain. A 23-amino-acid deletion (amino acids 402 to 424 of the ORF1a-encoded protein) largely abolished the inhibitory effect of nsp2 on ISG15 production and conjugation, but no viable recombinant virus was recovered. A 19-amino-acid deletion (amino acids 402 to 420), in combination with a downstream point mutation (S465A), partially relieved the ISG15 antagonist function and yielded a viable recombinant virus. Taken together, our data demonstrate that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.
New treatments for acute promyelocytic leukemia (APL) are needed. APL cell treatment with all-trans-retinoic acid (RA) degrades the chimeric, dominant negative-acting transcription factor PML/RARα, which is generated by chromosomal translocation. The E1-like ubiquitin-activating enzyme UBE1L associates with interferon stimulated gene ISG15 that binds and represses PML/RARα protein. Ubiquitin protease UBP43/USP18 removes ISG15 from conjugated proteins. In this study, we explored how RA regulates UBP43 expression and the effects of UBP43 on PML/RARα stability and APL growth, apoptosis and differentiation. RA treatment induced UBE1L, ISG15 and UBP43 expression in RA-sensitive but not RA-resistant APL cells. Similar in vivo findings were obtained in a transgenic mouse model of transplantable APL and in the RA response of leukemic cells harvested directly from APL patients. UBP43 knockdown repressed PML/RARα protein levels and inhibited RA-sensitive or RA-resistant APL cell growth by destabilizing the PML domain of PML/RARα. This inhibitory effect promoted apoptosis but did not affect the differentiation response in these APL cells. In contrast, elevation of UBP43 expression stabilized PML/RARα protein and inhibited apoptosis. Taken together, our findings define UBP43 as a novel candidate drug target for APL treatment.
UBP43/USP18; UBE1L; ISG15; acute promyelocytic leukemia; and PML/RARα
The anti-apoptotic protein FLIPS is a key suppressor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) -induced apoptosis in human glioblastoma multiforme (GBM) cells. We previously reported that a novel phosphatase and tensin homolog (PTEN)-Akt-atrophin interacting protein 4 (AIP4) pathway regulates FLIPS ubiquitination and stability, although the means by which PTEN and Akt were linked to AIP4 activity were unclear. We here report that a second regulator of ubiquitin metabolism, the ubiquitin-specific protease (USP) 8, is a downstream target of Akt, and that USP8 links Akt to AIP4 and the regulation of FLIPS stability and TRAIL resistance. In human GBM xenografts, levels of USP8 correlated inversely with pAkt levels, and genetic or pharmacologic manipulation of Akt regulated USP8 levels in an inverse manner. Over-expression of WT USP8, but not catalytically inactive USP8, increased FLIPS ubiquitination, decreased FLIPS half-life, decreased FLIPS steady-state levels, and decreased TRAIL resistance, while siRNA-mediated suppression of USP8 levels had the opposite effects. Because high levels of the USP8 deubiquitinase correlated with high levels of FLIPS ubiquitination, USP8 appeared to control FLIPS ubiquitination through an intermediate target. Consistent with this idea, over-expression of WT USP8 decreased ubiquitination of the FLIPS E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIPS interaction, while siRNA-mediated suppression of USP8 increased AIP4 ubiquitination. Furthermore, the suppression of FLIPS levels by USP8 over-expression was reversed by introduction of siRNA targeting AIP4. These results show that USP8, a downstream target of Akt, regulates the ability of AIP4 to control FLIPS stability and TRAIL sensitivity.
glioblastoma; TRAIL; ubiquitin; PTEN; USP8
New pharmacologic targets are needed for lung cancer. One candidate pathway to target is composed of the E1-like ubiquitin-activating enzyme (UBE1L) that associates with interferon-stimulated gene 15 (ISG15), which complexes with and destabilizes cyclin D1. Ubiquitin protease 43 (UBP43/USP18) removes ISG15 from conjugated proteins. This study reports that gain of UBP43 stabilized cyclin D1, but not other D-type cyclins or cyclin E. This depended on UBP43 enzymatic activity; an enzymatically inactive UBP43 did not affect cyclin D1 stability. As expected, small interfering RNAs (siRNAs) that reduced UBP43 expression also decreased cyclin D1 levels and increased apoptosis in a panel of lung cancer cell lines. Forced cyclin D1 expression rescued UBP43 apoptotic effects, which highlighted the importance of cyclin D1 in conferring this. Short hairpin RNA (shRNA)-mediated reduction of UBP43 significantly increased apoptosis and reduced murine lung cancer growth in vitro and in vivo after transplantation of these cells into syngeneic mice. These cells also exhibited increased response to all-trans-retinoic acid (RA), interferon (IFN), or cisplatin treatments. Notably, gain of UBP43 expression antagonized these effects. Normal-malignant human lung tissue arrays were examined independently for UBP43, cyclin D1, and cyclin E immunohistochemical expression. UBP43 was significantly (P < 0.01) increased in the malignant versus normal lung. A direct relationship was found between UBP43 and cyclin D1 (but not cyclin E) expression. Differential UBP43 expression was independently detected in a normal-malignant tissue array with diverse human cancers. Taken together, these findings uncovered UBP43 as a previously unrecognized anti-neoplastic target.
UBP43/USP18; cyclin D1; lung cancer
Inhibitor of DNA-binding-1 (ID1) transcription factor is essential for the proliferation and progression of many cancer types including leukemia. However, the ID1 protein has not yet been therapeutically targeted in leukemia. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a Ubiquitin-Rhodamine-based high throughput screening, we identified small molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the high nanomolar range. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth of leukemic cell lines in a dose dependent manner. A known USP1 inhibitor, Pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells. In addition, the growth of primary Acute Myeloid Leukemia (AML) patient-derived leukemic cells was inhibited by a USP1 inhibitor. Collectively, these results indicate that the novel small molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy.
USP1; ID1; small molecule inhibitors; leukemia
The chemokine receptor CXCR4 plays important roles in the immune and
nervous systems. Abnormal expression of CXCR4 contributes to cancer and
inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4
ubiquitination is known to accelerate CXCR4 degradation, little is known about
counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist,
induces a time-dependent association of USP14 with CXCR4, or its C terminus,
that is not mimicked by USP2A, USP4, or USP7, other members of the
deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is
time-dependent following CXCL12 stimulation. The physical interaction of CXCR4
and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor;
knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4
deubiquitination, whereas overexpression of USP14 promotes CXCR4
deubiquitination. We also observed that ubiquitination of CXCR4 facilitated
receptor degradation, whereas overexpression of USP14 or RNAi-induced
knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most
interestingly, CXCR4-mediated chemotactic cell migration was blocked by either
overexpression or RNAi-mediated knockdown of USP14, implying that a
CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated
state of the receptor, is critical for the ligand gradient sensing and
directed motility required for chemokine-mediated chemotaxis. Our observation
that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does
not mediate a chemotactic response to CXCL12, indicates the importance of this
covalent modification not only in marking receptors for degradation but also
for permitting CXCR4-mediated signaling. Finally, the indistinguishable
activation of ERK by wild typeor 3K/R-CXCR4 suggests that chemotaxis in
response to CXCL12 may be independent of the ERK cascade.
Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L−/− mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15−/− mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15−/− mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.
Type I interferon plays a critical role in the host defense to viral infection. Signaling through the type I IFN receptor allows for the induction of hundreds of interferon stimulated genes (ISGs) that generate an antiviral state within host cells. The ubiquitin-like molecule ISG15 has been shown to play an important role during multiple viral infections, including influenza virus infection. To date, the ability of ISG15 to protect against viral infection has been shown to be dependent on its ability to covalently bind (or conjugate) to target proteins, including the binding of viral proteins. We investigated the importance of the type I interferon response and ISG15 conjugation in a neonatal model of Chikungunya virus infection, a re-emerging human pathogen in the Indian Ocean region. Remarkably, the role of ISG15 during CHIKV infection appears to be conjugation independent, suggesting a non-classical role for ISG15 during viral infection. Our data also suggests that ISG15 plays an immunoregulatory role, as opposed to having direct antiviral function. Our CHIKV model may provide an opportunity to identify a novel mechanism by which ISG15 contributes to the innate immune response to viral infection.
Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.
Hepatitis C Virus (HCV) is a poor interferon (IFN) inducer, despite recognition of its RNA by the cytosolic RNA helicase RIG-I. This is due in part through cleavage of MAVS, a downstream adapter of RIG-I, by the HCV NS3/4A protease and through activation of the eIF2α-kinase PKR to control IFN translation. Here, we show that HCV also inhibits RIG-I activation through the ubiquitin-like protein ISG15 and that HCV triggers rapid induction of 49 genes, including ISG15, through a novel signaling pathway that precedes RIG-I and involves PKR as an adapter to recruit MAVS. Hence, we propose to divide the acute response to HCV infection into one early (PKR) and one late (RIG-I) phase, with the former controlling the latter. Furthermore, these data emphazise the need to check compounds designed as immune adjuvants for activation of the early acute phase before using them to sustain innate immunity.
Dermatomyositis (DM) is an autoimmune disease involving muscle and skin. Perifascicular atrophy (PFA) of myofibers is a specific and characteristic DM pathological lesion. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier with a poorly understood immunological role.
We generated microarray data measuring the expression of approximately 18,000 genes in each of 113 human muscle biopsy specimens. Biopsy specimens and cultured skeletal muscle were further studied using immunohistochemistry, immunoblotting, proteomic profiling by liquid chromatography/mass spectrometry, real-time quantitative PCR, and laser capture microdissection.
Transcripts encoding ISG15-conjugation pathway proteins were upregulated in DM with PFA (DM-PFA) muscle, with marked elevation of ISG15 (339-fold), HERC5 (62-fold), and USP18 (68-fold) present in all DM-PFA patients but none of 99 non-DM samples. Combined analysis with publicly available microarray datasets further showed marked ISG15 and USP18 transcript elevation had 100% sensitivity and specificity for 28 biopsies from adult DM-PFA and juvenile DM compared to 199 other muscle samples from a wide range of muscle diseases. Free ISG15 and ISG15-conjugated proteins were found by immunoblot only in DM-PFA muscle. Cultured human skeletal muscle exposed to type 1 interferons produced similar transcripts and both ISG15 protein and ISG15 conjugates. Laser capture microdissection followed by proteomic analysis showed deficiency of titin in DM perifascicular atrophic myofibers.
A large-scale microarray study of muscle samples from a diverse collection of muscle diseases revealed that the autoimmune disease dermatomyositis was uniquely associated with overactivation of the ISG15 conjugation pathway. Exposure of human skeletal muscle cell culture to type 1 interferons produces a molecular picture highly similar to that of human DM muscle biopsy specimens. Perifascicular atrophic myofibers in DM are deficient in a number of skeletal muscle proteins, most markedly titin.