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1.  Screen for ISG15-crossreactive Deubiquitinases 
PLoS ONE  2007;2(7):e679.
Background
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.
Results
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.
Conclusions
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.
doi:10.1371/journal.pone.0000679
PMCID: PMC1919423  PMID: 17653289
2.  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.
doi:10.1158/0008-5472.CAN-10-1100
PMCID: PMC2999664  PMID: 20935222
UBP43/USP18; UBE1L; ISG15; acute promyelocytic leukemia; and PML/RARα
3.  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.
doi:10.1128/MCB.25.24.11030-11034.2005
PMCID: PMC1316970  PMID: 16314524
4.  Ubp43 gene expression is required for normal Isg15 expression and fetal development 
Background
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.
Methods
Heterozygous (het) Ubp43 mice were mated and MM and CP implantation sites were collected on 12.5 and 17.5 days post-coitum (dpc).
Results
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.
Conclusion
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.
doi:10.1186/1477-7827-5-13
PMCID: PMC1852108  PMID: 17381847
5.  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.
doi:10.1158/1535-7163.MCT-12-0248
PMCID: PMC3438286  PMID: 22752428
UBP43/USP18; cyclin D1; lung cancer
6.  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.
doi:10.1128/MCB.25.15.6338-6345.2005
PMCID: PMC1190360  PMID: 16024773
7.  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.
doi:10.1371/journal.pone.0044884
PMCID: PMC3445607  PMID: 23028660
8.  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.
doi:10.4049/jimmunol.1101609
PMCID: PMC3345079  PMID: 22491252
Usp18; Ubp43; interferon; conventional dendritic cells; SOCS proteins; GM-CSF; pSTAT5
9.  Antiviral Properties of ISG15 
Viruses  2010;2(10):2154-2168.
The type I interferon system plays a critical role in limiting the spread of viral infection. Viruses induce the production of interferon (IFN), which after binding to the IFN-α/β receptor (IFNAR), and triggering of the JAK/STAT signaling cascade, results in the induction of interferon-stimulated genes (ISGs). These ISGs function to inhibit viral replication and to regulate the host immune response. Among these ISGs, the ubiquitin-like molecule, ISG15, is one of the most strongly induced proteins. Similar to ubiquitin, through an IFN induced conjugation cascade, ISG15 is covalently linked to a variety of cellular proteins, suggesting regulation of different cellular processes. Studies performed over the past several years have shown that ISG15 plays a central role in the host’s antiviral response against many viruses. Mice lacking ISG15 display increased susceptibility to multiple viruses. Furthermore, several viruses have developed immune evasion strategies that directly target the ISG15 pathway. Work is now underway to determine the mechanism by which ISG15 functions as an antiviral molecule, such that therapies targeting this pathway can be developed in the future.
doi:10.3390/v2102154
PMCID: PMC3185569  PMID: 21994614
ISG15; interferon; antiviral; ubiquitin-like molecule
10.  Regulation of Mitochondrial Morphology by USP30, a Deubiquitinating Enzyme Present in the Mitochondrial Outer Membrane 
Molecular Biology of the Cell  2008;19(5):1903-1911.
Recent studies have suggested that ubiquitination of mitochondrial proteins participates in regulating mitochondrial dynamics in mammalian cells, but it is unclear whether deubiquitination is involved in this process. Here, we identify human ubiquitin-specific protease 30 (USP30) as a deubiquitinating enzyme that is embedded in the mitochondrial outer membrane. Depletion of USP30 expression by RNA interference induced elongated and interconnected mitochondria, depending on the activities of the mitochondrial fusion factors mitofusins, without changing the expression levels of the key regulators for mitochondrial dynamics. Mitochondria were rescued from this abnormal phenotype by ectopic expression of USP30 in a manner dependent on its enzymatic activity. Our findings reveal that USP30 participates in the maintenance of mitochondrial morphology, a finding that provides new insight into the cellular function of deubiquitination.
doi:10.1091/mbc.E07-11-1103
PMCID: PMC2366858  PMID: 18287522
11.  Interferon-Stimulated Gene 15 and the Protein ISGylation System 
Interferon-stimulated gene 15 (ISG15) is one of the most upregulated genes upon Type I interferon treatment or pathogen infection. Its 17 kDa protein product, ISG15, was the first ubiquitin-like modifier identified, and is similar to a ubiquitin linear dimer. As ISG15 modifies proteins in a similar manner to ubiquitylation, protein conjugation by ISG15 is termed ISGylation. Some of the primary enzymes that promote ISGylation are also involved in ubiquitin conjugation. The process to remove ISG15 from its conjugated proteins, termed de-ISGylation, is performed by a cellular ISG15-specific protease, ubiquitin-specific proteases with molecular mass 43 kDa (UBP43)/ubiquitin-specific proteases 18. Relative to ubiquitin, the biological function of ISG15 is still poorly understood, but ISG15 appears to play important roles in various biological and cellular functions. Therefore, there is growing interest in ISG15, as the study of free ISG15 and functional consequences of ISGylation/de-ISGylation may identify useful therapeutic targets. This review highlights recent discoveries and remaining questions important to understanding the biological functions of ISG15.
doi:10.1089/jir.2010.0110
PMCID: PMC3021351  PMID: 21190487
12.  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.
doi:10.1128/JVI.06466-11
PMCID: PMC3302520  PMID: 22258253
13.  Knockdown of USP18 Increases Alpha 2a Interferon Signaling and Induction of Interferon-Stimulating Genes but Does Not Increase Antiviral Activity in Huh7 Cells▿ 
The current standard of care for hepatitis C virus (HCV) patients is cotreatment with human alpha interferon (IFN-α) and ribavirin. The host factor USP18 functions to regulate the interferon signaling pathway by acting as an off-switch. In order to understand whether the inhibition of USP18 represents a valid target for the enhancement of interferon treatment for chronic viral diseases, we have used a wide range of RNA interference (RNAi) reagents to suppress USP18 gene expression in Huh7 cell lines. We demonstrate that a USP18 knockdown results in IFN-α2a signaling (measured by increased IFN-stimulated response element [ISRE] reporter gene activity, 2′,5′-oligoadenylate synthetase [2-5 OAS] expression, and ISG15 induction) that is increased by ∼100-fold, whereas the antiviral (AV) potency in both the Huh7 HCV subgenomic replicon assay and the Huh7.5 HCV infectious virus assay increased by ∼3-fold. While the degree of the USP18 knockdown of USP18 elicited by the different RNAi reagents correlated with the enhancement of IFN-α2a signaling, it did not correlate with the enhancement of AV activity. The failure of increased IFN-α2a signaling to fully translate into increased AV potency was also observed for encephalomyocarditis virus (EMCV) assays using Huh7.5 cells. These data suggest that the IFN-mediated AV response in Huh7.5 cells has only a limited dependence on USP18 activity.
doi:10.1128/AAC.00644-11
PMCID: PMC3165365  PMID: 21709085
14.  RNA interference-mediated USP22 gene silencing promotes human brain glioma apoptosis and induces cell cycle arrest 
Oncology Letters  2013;5(4):1290-1294.
Ubiquitin-specific protease 22 (USP22) is a novel tumor stem cell marker that plays a key role in tumorigenesis and cell cycle progression. However, the effect of silencing the USP22 gene on human brain glioma cell growth is not well understood. In the present study, high gene expression of USP22 was identified in human brain glioma cells. In addition, RNA interference technology was used to silence USP22 gene expression in human brain glioma cells. Silencing the USP22 gene was found to effectively inhibit proliferation of human brain glioma cells, resulting in cell apoptosis and cell cycle arrest at the G2/M phase. USP22 silencing was also found to lead to reduced expression of cell cycle proteins, including CDK1, CDK2 and CyclinB1. In summary, in this study the USP22 gene was demonstrated to play a key regulatory role in the growth of human brain glioma cells by affecting progression of apoptosis and the cell cycle.
doi:10.3892/ol.2013.1188
PMCID: PMC3629196  PMID: 23599781
RNA interference; USP22 gene; human brain glioma; cell apoptosis; cell cycle
15.  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.
doi:10.1128/JVI.01294-12
PMCID: PMC3753986  PMID: 23824820
16.  Ubiquitin-specific protease 4 is inhibited by its ubiquitin-like domain 
EMBO Reports  2011;12(4):365-372.
Ubiquitin-specific protease 4 is inhibited by its ubiquitin-like domain
The ubiquitin-specific protease Usp4 contains an autoinhibitory Ubl domain in its catalytic domain. Inhibition can be relieved by interaction with a non-active USP, such as USP39.
USP4 is a member of the ubiquitin-specific protease (USP) family of deubiquitinating enzymes that has a role in spliceosome regulation. Here, we show that the crystal structure of the minimal catalytic domain of USP4 has the conserved USP-like fold with its typical ubiquitin-binding site. A ubiquitin-like (Ubl) domain inserted into the catalytic domain has autoregulatory function. This Ubl domain can bind to the catalytic domain and compete with the ubiquitin substrate, partially inhibiting USP4 activity against different substrates. Interestingly, other USPs, such as USP39, could relieve this inhibition.
doi:10.1038/embor.2011.33
PMCID: PMC3077250  PMID: 21415856
regulation; structure; Ubl; USP4
17.  Overexpression of the Ubiquitin-Specific Protease 7 Resulting from Transfection or Mutations in the ICP0 Binding Site Accelerates Rather than Depresses Herpes Simplex Virus 1 Gene Expression 
Journal of Virology  2012;86(23):12871-12878.
Earlier studies reported that ICP0, a key regulatory protein encoded by herpes simplex virus 1 (HSV-1), binds ubiquitin-specific protease 7 (USP7). The fundamental conclusion of these studies is that depletion of USP7 destabilized ICP0, that ICP0 mediated the degradation of USP7, and that amino acid substitutions in ICP0 that abolished binding to USP7 significantly impaired the ability of HSV-1 to replicate. We show here that, indeed, depletion of USP7 leads to reduction of ICP0 and that USP7 is degraded in an ICP0-dependent manner. However, overexpression of USP7 or substitution in ICP0 of a single amino acid to abolish binding to USP7 accelerated the accumulation of viral mRNAs and proteins at early times after infection and had no deleterious effect on virus yields. A clue as to why USP7 is degraded emerged from the observation that, notwithstanding the accelerated expression of viral genes, the plaques formed by the mutant virus were very small, implying a defect in virus transmission from cell to cell.
doi:10.1128/JVI.01981-12
PMCID: PMC3497670  PMID: 22993145
18.  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.
doi:10.1074/jbc.M808507200
PMCID: PMC2645827  PMID: 19106094
19.  UBE1L Causes Lung Cancer Growth Suppression by Targeting Cyclin D1 
Molecular cancer therapeutics  2008;7(12):3780-3788.
UBE1L is the E1-like ubiquitin-activating enzyme for the interferon-stimulated gene, 15 KDa protein (ISG15). The UBE1L-ISG15 pathway was previously proposed to target lung carcinogenesis by inhibiting cyclin D1 expression. This study extends prior work by reporting UBE1L promotes a complex between ISG15 and cyclin D1 and inhibited cyclin D1, but not other G1 cyclins. Transfection of the UBE1L-ISG15 deconjugase, ubiquitin specific protein 18 (UBP43), antagonized UBE1L-dependent inhibition of cyclin D1 and ISG15-cyclin D1 conjugation. A lysine-less cyclin D1 species was resistant to these effects. UBE1L transfection reduced cyclin D1 protein, but not mRNA expression. Cycloheximide (CHX) treatment augmented this cyclin D1 protein instability. UBE1L knock-down increased cyclin D1 protein. UBE1L was independently retrovirally transduced into human bronchial epithelial (HBE) and lung cancer cells. This reduced cyclin D1 expression and clonal cell growth. Treatment with the retinoid X receptor (RXR) agonist bexarotene induced UBE1L and reduced cyclin D1 immunoblot expression. A proof of principle bexarotene clinical trial was independently examined for UBE1L, ISG15, cyclin D1 and Ki-67 immunohistochemical expression profiles in pre- versus post-treatment tumor biopsies. Increased UBE1L with reduced cyclin D1 and Ki-67 expression occurred in human lung cancer when a therapeutic bexarotene intratumoral level was achieved. Thus, a mechanism for UBE1L-mediated growth suppression was found by UBE1LISG15 preferentially inhibited cyclin D1. Molecular therapeutic implications are discussed.
doi:10.1158/1535-7163.MCT-08-0753
PMCID: PMC2638083  PMID: 19074853
UBE1L; ISG15; cyclin D1; lung cancer; growth suppression
20.  RNA Interference Screen Identifies Usp18 as a Regulator of Epidermal Growth Factor Receptor Synthesis 
Molecular Biology of the Cell  2009;20(6):1833-1844.
Elevated expression of epidermal growth factor receptor (EGFR) contributes to the progression of many types of cancer. Therefore, we developed a high-throughput screen to identify proteins that regulate the levels of EGFR in squamous cell carcinoma. Knocking down various ubiquitination-related genes with small interfering RNAs led to the identification of several novel genes involved in this process. One of these genes, Usp18, is a member of the ubiquitin-specific protease family. We found that knockdown of Usp18 in several cell lines reduced expression levels of EGFR by 50–80%, whereas the levels of other receptor tyrosine kinases remained unchanged. Overexpression of Usp18 elevated EGFR levels in a manner requiring the catalytic cysteine of Usp18. Analysis of metabolically radiolabeled cells showed that the rate of EGFR protein synthesis was reduced up to fourfold in the absence of Usp18. Interestingly, this dramatic reduction occurred despite no change in the levels of EGFR mRNA. This suggests that depletion of Usp18 inhibited EGFR mRNA translation. In fact, this inhibition required the presence of native 5′ and 3′ untranslated region sequences on EGFR mRNA. Together, our data provide evidence for the novel mechanism of EGFR regulation at the translational step of receptor synthesis.
doi:10.1091/mbc.E08-08-0880
PMCID: PMC2655262  PMID: 19158387
21.  Ovarian Tumor (OTU)-domain Containing Viral Proteases Evade Ubiquitin- and ISG15-dependent Innate Immune Responses 
Cell host & microbe  2007;2(6):404-416.
Summary
Ubiquitin (Ub) and interferon stimulated gene product 15 (ISG15) reversibly conjugate to proteins via a conserved LRLRGG C-terminal motif, mediating important innate antiviral responses. The ovarian tumor (OTU) domain represents a superfamily of predicted proteases found in eukaryotic, bacterial and viral proteins, some of which have Ub-deconjugating activity. We show that the OTU domain-containing proteases of nairoviruses and arteriviruses hydrolyze Ub and ISG15 from cellular target proteins. This broad activity contrasts with the target specificity of known mammalian OTU domain-containing proteins. The biological significance of this activity of viral OTU domain-containing proteases was evidenced by their capacity to inhibit NF-κB dependent signaling and to antagonize the antiviral effects of ISG15 during Sindbis virus infection in vivo. The deconjugating activity of viral OTU proteases represents a novel viral immune evasion mechanism that inhibits Ub-and ISG15-dependent antiviral pathways.
doi:10.1016/j.chom.2007.09.014
PMCID: PMC2184509  PMID: 18078692
22.  Interferon-Induced ISG15 Conjugation Inhibits Influenza A Virus Gene Expression and Replication in Human Cells▿  
Journal of Virology  2009;83(12):5971-5977.
The ubiquitin-like ISG15 protein, as well as its conjugating enzymes, is induced by type I interferons (IFNs). Experiments using ISG15 knockout (ISG15−/−) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus. However, in contrast to the virus inhibition results for mice, the rates of virus replication in ISG15+/+ and ISG15−/− mouse embryo fibroblasts in tissue culture were similar. Here we focus on human tissue culture cells and on the effect of ISG15 and/or its conjugation on influenza A virus gene expression and replication in such cells. We demonstrate that IFN-induced antiviral activity against influenza A virus in human cells is significantly alleviated by inhibiting ISG15 conjugation using small interfering RNAs directed against ISG15-conjugating enzymes. IFN-induced antiviral activity against influenza A virus protein synthesis was reduced 5- to 20-fold by suppressing ISG15 conjugation. The amounts of the viral proteins that were restored by these siRNA treatments were approximately 40 to 50% of the amounts produced in cells that were not pretreated with IFN. Further, we show that ISG15 conjugation inhibits influenza A virus replication 10- to 20-fold at early times after infection in human cells. These results show that ISG15 conjugation plays a substantial role in the antiviral state induced by IFN in human cells. In contrast, we show that in mouse embryo fibroblasts ISG15 conjugation not only does not affect influenza A virus replication but also does not contribute to the IFN-induced antiviral activity against influenza A virus gene expression.
doi:10.1128/JVI.01667-08
PMCID: PMC2687383  PMID: 19357168
23.  Hyaluronan- and RNA-binding deubiquitinating enzymes of USP17 family members associated with cell viability 
BMC Genomics  2006;7:292.
Background
Protein degradation by the ubiquitin system plays a crucial role in numerous cellular signaling pathways. Deubiquitination, a reversal of ubiquitination, has been recognized as an important regulatory step in the ubiquitin-dependent degradation pathway.
Results
While identifying putative ubiquitin specific protease (USP) enzymes that contain a conserved Asp (I) domain in humans, 4 USP17 subfamily members, highly homologous to DUB-3, have been found (USP17K, USP17L, USP17M, and USP17N), from human chorionic villi. Expression analysis showed that USP17 transcripts are highly expressed in the heart, liver, and pancreas and are expressed moderately in various human cancerous cell lines. Amino acid sequence analysis revealed that they contain the highly conserved Cys, His, and Asp domains which are responsible for the deubiquitinating activity. Biochemical enzyme assays indicated that they have deubiquitinating activity. Interestingly, the sequence analysis showed that these proteins, with exception of USP17N, contain the putative hyaluronan/RNA binding motifs, and cetylpyridinium chloride (CPC)-precipitation analysis confirmed the association between these proteins and intracellular hyaluronan and RNA.
Conclusion
Here, we report that the overexpression of these proteins, with exception of USP17N, leads to apoptosis, suggesting that the hyaluronan and RNA binding motifs in these enzymes play an important role in regulating signal transduction involved in cell death.
doi:10.1186/1471-2164-7-292
PMCID: PMC1665497  PMID: 17109758
24.  Ube1L and Protein ISGylation Are Not Essential for Alpha/Beta Interferon Signaling 
Molecular and Cellular Biology  2006;26(2):472-479.
The expression of ubiquitin-like modifier ISG15 and its conjugation to target proteins are highly induced by interferon (IFN) stimulation and during viral and bacterial infections. However, the biological significance of this modification has not been clearly understood. To investigate the function of protein modification by ISG15, we generated a mouse model deficient in UBE1L, an ISG15-activating enzyme. Ube1L−/− mice did not produce ISG15 conjugates but expressed free ISG15 normally. ISGylation has been implicated in the reproduction and innate immunity. However, Ube1L−/− mice were fertile and exhibited normal antiviral responses against vesicular stomatitis virus and lymphocytic choriomeningitis virus infection. Our results indicate that UBE1L and protein ISGylation are not critical for IFN-α/β signaling via JAK/STAT activation. Moreover, using Ube1L/Ubp43 double-deficient mice, we showed that lack of UBP43, but not the increase of protein ISGylation, is related to the increased IFN signaling in Ubp43-deficient mice.
doi:10.1128/MCB.26.2.472-479.2006
PMCID: PMC1346917  PMID: 16382139
25.  ISG15 Modulates Development of the Erythroid Lineage 
PLoS ONE  2011;6(10):e26068.
Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15-/- bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation.
doi:10.1371/journal.pone.0026068
PMCID: PMC3192153  PMID: 22022510

Results 1-25 (940486)