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1.  Screen for ISG15-crossreactive Deubiquitinases 
PLoS ONE  2007;2(7):e679.
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.
PMCID: PMC1919423  PMID: 17653289
2.  Protein Interferon-Stimulated Gene 15 Conjugation Delays but Does Not Overcome Coronavirus Proliferation in a Model of Fulminant Hepatitis 
Journal of Virology  2014;88(11):6195-6204.
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.
PMCID: PMC4093886  PMID: 24648452
3.  Cynoglossus semilaevis ISG15: A Secreted Cytokine-Like Protein That Stimulates Antiviral Immune Response in a LRGG Motif-Dependent Manner 
PLoS ONE  2012;7(9):e44884.
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.
PMCID: PMC3445607  PMID: 23028660
4.  Ubp43 gene expression is required for normal Isg15 expression and fetal development 
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.
PMCID: PMC1852108  PMID: 17381847
5.  ISG15, an Interferon-Stimulated Ubiquitin-Like Protein, Is Not Essential for STAT1 Signaling and Responses against Vesicular Stomatitis and Lymphocytic Choriomeningitis Virus 
Molecular and Cellular Biology  2005;25(15):6338-6345.
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.
PMCID: PMC1190360  PMID: 16024773
6.  Reexamination of the Role of Ubiquitin-Like Modifier ISG15 in the Phenotype of UBP43-Deficient Mice 
Molecular and Cellular Biology  2005;25(24):11030-11034.
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.
PMCID: PMC1316970  PMID: 16314524
7.  Ubiquitin-specific proteases are differentially expressed throughout the Schistosoma mansoni life cycle 
Parasites & Vectors  2015;8:349.
The ubiquitination process can be reversed by deubiquitinating enzymes (DUBs). These proteases are involved in ubiquitin processing, in the recovery of modified ubiquitin trapped in inactive forms, and in the recycling of ubiquitin monomers from polyubiquitinated chains. The diversity of DUB functions is illustrated by their number and variety of their catalytic domains with specific 3D architectures. DUBs can be divided into five subclasses: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs or UBPs), ovarian tumour proteases (OTUs), Machado-Joseph disease proteases (MJDs) and JAB1/MPN/Mov34 metalloenzymes (JAMMs).
Considering the role that the ubiquitin-proteasome system has been shown to play during the development of Schistosoma mansoni, our main goal was to identify and characterize SmUSPs. Here, we showed the identification of putative ubiquitin-specific proteases using bioinformatic approaches. We also evaluated the gene expression profile of representative USP family members using qRT-PCR.
We reported 17 USP family members in S. mansoni that present a conservation of UCH domains. Furthermore, the putative SmUSP transcripts analysed were detected in all investigated stages, showing distinct expression during S. mansoni development. The SmUSPs exhibiting high expression profiles were SmUSP7, SmUSP8, SmUSP9x and SmUSP24.
S. mansoni USPs showed changes in expression levels for different life cycle stages indicating their involvement in cellular processes required for S. mansoni development. These data will serve as a basis for future functional studies of USPs in this parasite.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-015-0957-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4485857  PMID: 26112833
Ubiquitination; Deubiquitination; Deubiquitinating enzymes (DUBs); Differential expression; Schistosome development
8.  Specific and Covalent Targeting of Conjugating and Deconjugating Enzymes of Ubiquitin-Like Proteins 
Molecular and Cellular Biology  2004;24(1):84-95.
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.
PMCID: PMC303361  PMID: 14673145
9.  Ubiquitin-specific protease 14 regulates c-Jun N-terminal kinase signaling at the neuromuscular junction 
Ubiquitin-specific protease 14 (USP14) is one of three proteasome-associated deubiquitinating enzymes that remove ubiquitin from proteasomal substrates prior to their degradation. In vitro evidence suggests that inhibiting USP14’s catalytic activity alters the turnover of ubiquitinated proteins by the proteasome, although whether protein degradation is accelerated or delayed seems to be cell-type and substrate specific. For example, combined inhibition of USP14 and the proteasomal deubiquitinating enzyme UCH37 halts protein degradation and promotes apoptosis in multiple myeloma cells, whereas USP14 inhibition alone accelerates the degradation of aggregate-prone proteins in immortalized cell lines. These findings have prompted interest in USP14 as a therapeutic target both inside and outside of the nervous system. However, loss of USP14 in the spontaneously occurring ataxia mouse mutant leads to a dramatic neuromuscular phenotype and early perinatal lethality, suggesting that USP14 inhibition may have adverse consequences in the nervous system. We therefore expressed a catalytically inactive USP14 mutant in the mouse nervous system to determine whether USP14’s catalytic activity is required for neuromuscular junction (NMJ) structure and function.
Mice expressing catalytically inactive USP14 in the nervous system exhibited motor deficits, altered NMJ structure, and synaptic transmission deficits that were similar to what is observed in the USP14-deficient ataxia mice. Acute pharmacological inhibition of USP14 in wild type mice also reduced NMJ synaptic transmission. However, there was no evidence of altered proteasome activity when USP14 was inhibited either genetically or pharmacologically. Instead, these manipulations increased the levels of non-proteasome targeting ubiquitin conjugates. Specifically, we observed enhanced proteasome-independent ubiquitination of mixed lineage kinase 3 (MLK3). Consistent with the direct activation of MLK3 by ubiquitination, we also observed increased activation of its downstrea targets MAP kinase kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK). In vivo inhibition of JNK improved motor function and synapse structure in the USP14 catalytic mutant mice.
USP14’s catalytic activity is required for nervous system structure and function and has an ongoing role in NMJ synaptic transmission. By regulating the ubiquitination status of protein kinases, USP14 can coordinate the activity of intracellular signaling pathways that control the development and activity of the NMJ.
Electronic supplementary material
The online version of this article (doi:10.1186/1750-1326-10-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4417291  PMID: 25575639
USP14; JNK; Ubiquitin; Neuromuscular junction; MLK3; Synapse; Ubiquitin proteasome system; K63-linked ubiquitin; Motor neuron; Motor endplate disease
10.  Vaccinia Virus E3 Protein Prevents the Antiviral Action of ISG15 
PLoS Pathogens  2008;4(7):e1000096.
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.
Author Summary
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.
PMCID: PMC2434199  PMID: 18604270
11.  Usp18 promotes conventional CD11b+ dendritic cell development 
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.
PMCID: PMC3345079  PMID: 22491252
Usp18; Ubp43; interferon; conventional dendritic cells; SOCS proteins; GM-CSF; pSTAT5
12.  Zebrafish ISG15 Exerts a Strong Antiviral Activity against RNA and DNA Viruses and Regulates the Interferon Response 
Journal of Virology  2013;87(18):10025-10036.
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.
PMCID: PMC3753986  PMID: 23824820
13.  Structural Basis for the Ubiquitin-Linkage Specificity and deISGylating Activity of SARS-CoV Papain-Like Protease 
PLoS Pathogens  2014;10(5):e1004113.
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.
Author Summary
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.
PMCID: PMC4031219  PMID: 24854014
14.  Coordinated Regulation and Widespread Cellular Expression of Interferon-Stimulated Genes (ISG) ISG-49, ISG-54, and ISG-56 in the Central Nervous System after Infection with Distinct Viruses▿  
Journal of Virology  2006;81(2):860-871.
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.
PMCID: PMC1797448  PMID: 17079283
15.  Human intracellular ISG15 prevents interferon-α/β over-amplification and auto-inflammation 
Nature  2014;517(7532):89-93.
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.
PMCID: PMC4303590  PMID: 25307056
16.  Contribution of increased ISG15, ISGylation and deregulated Type I IFN signaling in Usp18 mutant mice during the course of bacterial infections 
Genes and immunity  2014;15(5):282-292.
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.
PMCID: PMC4111656  PMID: 24807690 CAMSID: cams4329
USP18; innate immunity; Salmonella; mycobacteria; type I IFN; ISGylation; autophagy
17.  Blockade of the Ubiquitin Protease UBP43 Destabilizes the Transcription Factor PML/RARα and Inhibits Growth of Acute Promyelocytic Leukemia 
Cancer research  2010;70(23):9875-9885.
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.
PMCID: PMC2999664  PMID: 20935222
UBP43/USP18; UBE1L; ISG15; acute promyelocytic leukemia; and PML/RARα
18.  Nonstructural Protein 2 of Porcine Reproductive and Respiratory Syndrome Virus Inhibits the Antiviral Function of Interferon-Stimulated Gene 15 
Journal of Virology  2012;86(7):3839-3850.
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.
PMCID: PMC3302520  PMID: 22258253
19.  UBE1L represses PML/RARα by targeting the PML domain for ISG15ylation 
Molecular cancer therapeutics  2008;7(4):905-914.
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.
PMCID: PMC2597092  PMID: 18413804
20.  A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation 
PLoS Pathogens  2013;9(3):e1003273.
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.
Author Summary
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.
PMCID: PMC3610741  PMID: 23555268
21.  USP8 links the PTEN-Akt-AIP4 pathway to the control of FLIPS stability and TRAIL sensitivity in glioblastoma multiforme 
Cancer research  2010;70(12):5046-5053.
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.
PMCID: PMC2891229  PMID: 20484045
glioblastoma; TRAIL; ubiquitin; PTEN; USP8
22.  Evidence for the Ubiquitin Protease UBP43 as an Antineoplastic Target 
Molecular cancer therapeutics  2012;11(9):1968-1977.
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.
PMCID: PMC3438286  PMID: 22752428
UBP43/USP18; cyclin D1; lung cancer
23.  Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation* 
The Journal of Biological Chemistry  2009;284(9):5742-5752.
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.
PMCID: PMC2645827  PMID: 19106094
24.  ISG15 Is Critical in the Control of Chikungunya Virus Infection Independent of UbE1L Mediated Conjugation 
PLoS Pathogens  2011;7(10):e1002322.
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.
Author Summary
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.
PMCID: PMC3197620  PMID: 22028657
25.  Interferon-Stimulated Gene 15 (ISG15) Conjugates Proteins in Dermatomyositis Muscle with Perifascicular Atrophy 
Annals of neurology  2010;67(1):53-63.
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.
PMCID: PMC2875060  PMID: 20186858

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