USP18 Data Analysis

HGNC Gene Name: ubiquitin specific peptidase 18
HGNC Gene Symbol: USP18
Identifiers: hgnc:12616 NCBIGene:11274 uniprot:Q9UMW8
Orthologs: rgd:1359153
INDRA Statements: deubiquitinations all statements
Pathway Commons: Search for USP18
Number of Papers: 328 retrieved on 2023-02-19

DepMap Analysis

The Dependency Map (DepMap) is a genome-wide pooled CRISPR-Cas9 knockout proliferation screen conducted in more than 700 cancer cell lines spanning many different tumor lineages. Each cell line in the DepMap contains a unique barcode, and each gene knockout is assigned a “dependency score” on a per cell-line basis which quantifies the rate of CRISPR-Cas9 guide drop. It has been found that proteins with similar DepMap scores across cell lines, a phenomenon known as co-dependent genes, have closely related biological functions. This can include activity in the same or parallel pathways or membership in the same protein complex or the same pathway.

We identified the strongest seven co-dependent genes (“Symbol”) for DUBs and ran GO enrichment analysis. We used Biogrid, IntAct, and Pathway Commons PPIDs, and the NURSA protein-protein interaction databases (PPIDs) to determine whether co-dependent genes interact with one another. The “Evidence” column contains the PPIDs in which the interaction appears as well as whether there is support for the association by an INDRA statement. As another approach to identify potential interactors, we looked at proteomics data from the Broad Institute's Cancer Cell Line Encyclopedia (CCLE) for proteins whose expression across ~375 cell lines strongly correlated with the abundance of each DUB; it has previously been observed that proteins in the same complex are frequently significantly co-expressed. The correlations and associated p-values in the CCLE proteomics dataset are provided. And, we determined whether co-dependent genes yield similar transcriptomic signatures in the Broad Institute's Connectivity Map (CMap). A CMap score greater than 90 is considered significantly similar.

DepMap Correlations

Symbol	Name	DepMap Correlation	Evidence
ISG15	ISG15 ubiquitin like modifier	0.405	BioGRID IntAct Pathway Commons INDRA (20) Reactome (7)
IFNAR1	interferon alpha and beta receptor subunit 1	-0.395	INDRA (7) Reactome (5)
IFNAR2	interferon alpha and beta receptor subunit 2	-0.381	BioGRID IntAct Pathway Commons INDRA (9) Reactome (5)
IRF9	interferon regulatory factor 9	-0.372	INDRA (2) Reactome (4)
STAT2	signal transducer and activator of transcription 2	-0.346	BioGRID Pathway Commons INDRA (10) Reactome (6)
TYK2	tyrosine kinase 2	-0.318	Reactome (6)
ZNF74	zinc finger protein 74	0.308

Dependency GO Term Enrichment

Gene set enrichment analysis was done on the genes correlated with USP18using the terms from Gene Ontology and gene sets derived from the Gene Ontology Annotations database via MSigDB.

Using the biological processes and other Gene Ontology terms from well characterized DUBs as a positive control, several gene set enrichment analyses were considered. Threshold-less methods like GSEA had relatively poor results. Over-representation analysis with a threshold of of the top 7 highest absolute value Dependency Map correlations yielded the best results and is reported below.

GO Identifier	GO Name	GO Type	p-value	p-value (adj.)	q-value
GO:0034340	response to type I interferon	Biological Process	9.98e-14	7.49e-12	3.26e-12
GO:0009615	response to virus	Biological Process	2.83e-08	2.12e-06	4.62e-07
GO:0051607	defense response to virus	Biological Process	8.34e-07	6.26e-05	9.07e-06
GO:0004904	interferon receptor activity	Molecular Function	1.17e-06	8.75e-05	9.51e-06
GO:0097696	receptor signaling pathway via STAT	Biological Process	2.12e-05	1.59e-03	1.20e-04
GO:0035455	response to interferon-alpha	Biological Process	2.21e-05	1.66e-03	1.20e-04
GO:0004896	cytokine receptor activity	Molecular Function	5.12e-04	3.84e-02	2.39e-03

Transcriptomics

The following table shows the significantly differentially expressed genes after knocking out USP18 using CRISPR-Cas9.

Knockout Differential Expression

Symbol	Name	log₂-fold-change	p-value	p-value (adj.)
VIM	vimentin	6.37e-01	1.06e-10	1.39e-06
RPL14	ribosomal protein L14	5.81e-01	1.43e-08	9.37e-05
TOP1	DNA topoisomerase I	6.44e-01	3.65e-07	1.59e-03
HSPA8	heat shock protein family A (Hsp70) member 8	4.24e-01	6.34e-07	2.07e-03
RPS2	ribosomal protein S2	5.77e-01	1.05e-06	2.74e-03
RPL7A	ribosomal protein L7a	7.29e-01	3.92e-06	8.55e-03
ADCY6	adenylate cyclase 6	8.89e-01	1.58e-05	2.70e-02
WDR82	WD repeat domain 82	6.61e-01	1.65e-05	2.70e-02
TEX261	testis expressed 261	8.06e-01	3.34e-05	4.86e-02

Gene Set Enrichment Analysis

There were too few differentially expressed genes to run a meaningful GSEA.

Literature Mining

INDRA was used to automatically assemble known mechanisms related to USP18 from literature and knowledge bases. The first section shows only DUB activity and the second shows all other results.

Deubiquitinase Activity

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USP18 deubiquitinates MAP3K7. 10 / 20

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Another way that USP18 inhibited NF-κB activation is by deubiquitinating K63-Ub of TAK1 and NEMO [42].

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USP18 deubiquitinates TAK1 in a protease dependent manner in HEK293 cells.

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In contrast, they suggest that USP18 inhibits ubiquitination of the TAK1 and TAB1 complex in a protease dependent manner XREF_BIBR, XREF_BIBR.

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These observations suggest that USP18 and USP19 deubiquitinate TAK1 in a cell type specific dependent manner.

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In contrast, they suggest that USP18 inhibits ubiquitination of the TAK1/TAB1 complex in a protease dependent manner30,31.

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USP18 can inhibit the ubiquitination of the TAK1 and TAB complex, thereby inhibiting IL-2 production and promoting IL-17 production and synthesis.

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USP18 directly cleaves the K63 linked polyubiquitin chains, but not K48 linked polyubiquitin chains from TAK1 in a protease dependent manner since the USP18 catalytically inactive mutant can not deubiquitinate TAK1.

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USP18 binds to and inhibits ubiquitination of the TAK1 and TAB complex, thereby restricting IL-2 production and promoting IL-17 production.

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Collectively, these data suggest that USP18 targets the TAB1 and TAK1 complex and inhibits TAK1 polyubiquitination modification and kinase activity, thereby restricting TCR mediated NF-kappaB and NFAT activation and subsequent expression of IL-2.

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Previous studies showed that USP18 potently abolishes the polyubiquitination of TAK1 and TAB1 complex XREF_BIBR.

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USP18 deubiquitinates IKBKG. 10 / 11

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To investigate the precise mechanisms mediating inhibition of NEMO ubiquitination by USP18, we generated NEMO deletion mutants (XREF_FIG), containing a domain responsible for IKK binding, TANK binding, UBAN, or IkappaBalpha binding.

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Collectively, our findings revealed that USP18 inhibits TAK1 and NEMO ubiquitination through different mechanisms.

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On the other hand, USP18 inhibits NEMO ubiquitination by directly binding to its UBAN domain.

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USP18 negatively regulates NF-κB signaling by targeting TAK1 and NEMO for deubiquitination through distinct mechanisms

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These results indicate that USP18 inhibits NEMO ubiquitination as well as NF-kappaB activity in a protease independent manner.

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Another way that USP18 inhibited NF-κB activation is by deubiquitinating K63-Ub of TAK1 and NEMO [42] .

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USP18 inhibits NEMO ubiquitination at the Lys -325 and 326 sites by masking the UBAN domain of NEMO.

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First, we used the two-step immunoprecipitation assay to assess whether USP18 prevents the conjugated ubiquitination of NEMO.

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Since the UBAN domain of NEMO contains all five K63 linked ubiquitination sites (Lys 285, Lys 321, Lys 325, Lys 326, and Lys 399) XREF_BIBR and two linear ubiquitination sites for NEMO (Lys 285 and Lys 309) XREF_BIBR, USP18 may prevent NEMO ubiquitination by masking these ubiquitination sites through direct binding.

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We next tested whether USP18 inhibited NEMO ubiquitination through USP18 protease activity.

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USP18 deubiquitinates TAB1. 6 / 6

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To further examine whether USP18 deubiquitinates the TAK1 and TAB1 complex, we purified Flag tagged USP18 from 293T cells transiently transfected with Flag-USP18 plasmid by immunoprecipitation with anti-Flag agarose and elution with Flag peptide.

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Importantly, USP18 is associated with and deubiquitinates the TAK1 and TAB1 complex, thereby restricting expression of IL-2.

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In contrast, they suggest that USP18 inhibits ubiquitination of the TAK1 and TAB1 complex in a protease dependent manner XREF_BIBR, XREF_BIBR.

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In contrast, they suggest that USP18 inhibits ubiquitination of the TAK1/TAB1 complex in a protease dependent manner30,31.

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USP18 is associated with and deubiquitinates the TAK1-TAB1 complex

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Previous studies showed that USP18 potently abolishes the polyubiquitination of TAK1 and TAB1 complex XREF_BIBR.

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USP18 deubiquitinates STING1. 4 / 4

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USP18 does not deubiquitinate STING in vitro but facilitates USP20 to catalyze deubiquitination of STING in a manner independently of the enzymatic activity of USP18 (91).

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USP18 did not deubiquitinate STING in vitro but facilitated USP20 to catalyze deubiquitination of STING in a manner independent of the enzymatic activity of USP18.

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Subsequently, they searched for DUBs that interact with USP18 and found that knockdown of USP20 inhibited USP18 induced deubiquitination of STING and knockdown of USP18 inhibited USP20 induced deubiquitination of STING [XREF_BIBR].

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These results together supported that although USP18 does not deubiquitinate STING itself, USP18 recruits USP20 to deubiquitinate STING to suppress IFN synthesis [41].

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USP18 deubiquitinates TWIST1. 2 / 2

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USP18 deubiquitinates and stabilizes Twist1 to promote epithelial-mesenchymal transition in glioblastoma cells.

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USP18 affects Interferon

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USP18 inhibits Interferon.

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USP18 inhibits Interferon. 10 / 58

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USP18 down-regulates type I interferon signaling by blocking the access of Janus associated kinase 1 (JAK1) to the type I interferon receptor.

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For instance, human ubiquitin specific peptidase 18 (USP18) negatively regulates type-I IFN signaling by binding to the IFN receptor.

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USP18 is thought to inhibit the effect of IFN therapy by reducing its disponibility.

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As we and others have previously demonstrated that the ISG product USP18 suppresses IFN signaling as a form of negative feedback [XREF_BIBR, XREF_BIBR], reduced ISG mRNA translation under reduced nutrient conditions will be accompanied by sustained transcriptional induction of ISGs, resulting in a prolonged IFN response.

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USP11 upregulates IkappaB kinase alpha in a ubiquitin independent manner XREF_BIBR, while USP18 has been shown to negatively regulate interferon signalling through protein interactions that are independent of its isopeptidase activity XREF_BIBR.

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Recent data by Zhang and coworkers revealed, however, that USP18 attenuates JAK-STAT signaling, and thereby the type 1 IFN response, in a non enzymatic manner, i.e., by directly competing with JAK1 for binding to the IFNAR2 subunit of the type 1 IFN receptor.

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These observations established the key role of STAT2 in USP18 mediated inhibition of IFN signaling and moreover suggest that the increased STAT2 levels induced by IFN signaling may further enhance negative feedback by USP18.

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In addition to its deISGylase activity, USP18 also negatively regulates IFN signaling through association with the IFN receptor.

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The siRNA knockdown of USP18 in human cells consistently potentiated the ability of IFN to inhibit HCV-RNA replication and infectious virus particle production by a factor of 1-2 log (10).

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Besides being an active enzyme, USP18 negatively regulates type I interferon signalling independent of its protease activity [22] (Figure 1).

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USP18 bound to IFNAR2 inhibits Interferon. 2 / 2

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USP18 directly binds to IFN-alpha and beta receptor 2 (IFNAR2) in human KT-1 cells and, by competing for JAK1 binding, inhibits type I IFN signaling.

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Indeed, USP18 binds to IFNAR2 to inhibit type I IFN signaling and subsequently disrupting IFNAR2-JAK1 interaction, thus negatively regulating IFN signaling cascades [XREF_BIBR].

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USP18 bound to JAK1 inhibits Interferon. 2 / 2

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Based on mutational studies it was suggested that USP18 binds to the intracellular region of type I IFN receptor subunit IFNAR2 and outcompetes the downstream kinase JAK1 thereby abrogating IFN signaling36.

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USP18 bound to IFNA inhibits Interferon. 1 / 1

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The binding of USP18 with the IFN-α receptor has been shown to inhibit the interaction of STAT-1 with the IFN-α receptor and thus block downstream IFN signaling (12, 15).

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USP18 activates Interferon.

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USP18 activates Interferon. 10 / 27

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To ascertain that this enhanced type 1 IFN response with up-regulation of ISG expression was indeed caused by USP18 deficiency, we transduced USP18 deficient fibroblasts from P1 and P2 and control fibroblasts with lentiviral particles (LV)-expressing luciferase (Luc) or WT USP18.

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We have previously shown that USP18 can modulate the type 1 IFN response.

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PROTACS targeting USP18 might represent an interesting approach to specifically degrade USP18 and thus enhance type I IFN signaling.Beside direct destabilization, targeting the interaction of USP18 with important proteins such as STAT2 or ISG15 might constitute an option to interfere with its function.A sophisticated technique to directly study protein–protein interaction within a cellular context is the BRET (Bioluminescence Resonance Energy Transfer) assay where dipole-dipole energy is transferred from a luciferase to a fluorophore.

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The role of Usp18 mediated de-ISGylation in IFN stimulated cells -- when it occurs and what happens if it does not occur -- remains uncertain, and these questions are complicated by the fact that Usp18 has a second function in innate immune responses unrelated to deconjugation.

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ISG15 has also been shown to regulate type I interferon signalling by stabilizing USP18 (ref.4) and by interacting with leucine-rich repeat-containing protein 25 (LRRC25) to mediate the autophagic degradation of retinoic acid-inducible gene I protein (RIG-I; also known as DDX58)41.

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The knock-down of USP18 enhanced the ability of IFN to inhibit HCV replication in vitro.

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In individuals carrying the unfavorable allele, a high basal level of IFN-λ, ISGs, and USP18 will lead to a refractory state and unresponsiveness to the IFN-α treatment and failure to clear the infection.Figure 2: Interferon (IFN)-λ modulation of hematopoietic cells.

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Further investigation with the KEGG pathway enrichment analysis showed those up-regulated genes could cause the activation of the IFN-induced pathway, type II interferon signaling pathway, and regulation of protein ISGylation by the ISG15 deconjugating enzyme USP18 pathway (Figure 4B).

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ISG15 has also been shown to regulate type I interferon signalling by stabilizing USP18 (ref.

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Influenza B has evolved a mechanism to directly neutralize ISG15 with its NS1 protein24 and coronaviruses have a papain‐like protease that has deISGylase activity as a strategy to overcome ISG15,25, 26 indicating the importance of ISG15 in the antiviral response.In addition to its enzymatic activity, USP18 negatively regulates T1 IFN signaling.27 USP18 is recruited by STAT2 to the type I IFN receptor subunit, IFNAR2, where it binds to IFNAR2 and prevents phosphorylation of JAK1 by blocking the interaction of JAK1 and the IFNAR2 subunit.27, 28, 29 USP18 expression also plays a role in limiting TRAIL‐induced apoptosis and has also been shown to regulate the susceptibility of certain cancer cells to IFN‐α and drug‐induced apoptosis.30, 31Macrophages play an important role in HIV‐1 as reservoirs and can contribute directly to HIV‐1 pathogenesis.32 HIV‐1 in the ART era can be seen as a chronic disease characterized by chronic immune activation and chronic inflammation with a higher risk of non‐AIDS‐related morbidities and mortalities.

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USP18 bound to Interferon activates Interferon. 1 / 1

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Since we have shown that binding of Usp18 to type I IFN receptor subunit 2 (Ifnar2) is important for Usp18 mediated downregulation of type I IFN signalling, we investigated a potential interaction of Usp18 with the IFN-lambda specific receptor subunit IL-28R1.

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USP18 increases the amount of Interferon.

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USP18 increases the amount of Interferon. 2 / 2

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USP18 upregulates the expression and production of type I interferon following infection with Sendai virus (SeV) or Encephalomyocarditis virus (EMCV).

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The IFN signal is controlled by the negative regulator USP18, limiting the expression of interferon-stimulated genes (ISGs).

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Interferon affects USP18

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Interferon activates USP18.

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Interferon activates USP18. 10 / 53

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Finally, we assessed IFN induced USP18 accumulation in cells silenced for SKP2.

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Moreover, interferon-alpha increased the expression of interferon stimulated gene 15 (ISG15), ubiquitin specific peptidase 18 (USP18), and interleukin-6 (IL-6) via activation of STAT1.

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In contrast, in those cells that progressed beyond this phase, the IFN treatment could not initiate USP18 induction until the next cell cycle, resulting in extended delay times proportional to the times needed to reach the next cell cycle.

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Model fitting results and the parameter analysis.Pulsatile IFN-a treatment induces higher ISG expression in single cells.Heterogeneous delays in USP18 upregulation by IFN were observed in single cells.

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The inductions of un phosphorylated IFN stimulated gene factor 3 (U-ISGF3) -associated ISGs, IFN stimulated gene 15 (ISG15) and ubiquitin specific protease 18 (USP18), were also evaluated.

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Yet, the amount of ISGylated SKP2 is rather small, even in a context where global ISGylation is high as in IFN stimulated USP18 deficient fibroblasts.

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The IFN induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1.

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The increased expression of dsRNA receptor TLR3 and IFN induced genes ISG56, ISG43, Mx1 and IFIT3 after stimulation with poly I : C mimicking a viral infection indicates that these cell lines can be used as effective in vitro models to study the bat 's innate immune responses to virus infection XREF_BIBR, XREF_BIBR.

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The expression of USP18 is strongly induced by type I and type III interferons [ 2-5 ] , by the Toll-like receptor ( TLR ) agonists LPS [ 6-8 ] and polyI :C [ 6 ] ( synthetic analogy of dsRNA ) and by TNFalpha [ 7 ] .

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A keynote presentation by Glenn Randall (University of Chicago, USA) summarized the current understanding of the mechanisms by which interferon (IFN) controls HCV infection and described how 2 key IFN stimulated genes (ISGs), ISG15 and USP18, function within a protein modification cycle to regulate hepatic innate immunity and virus infection.

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Interferon activates mutated USP18. 1 / 1

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Contribution of increased ISG15, ISGylation and deregulated Type I IFN signaling in Usp18 mutant mice during the course of bacterial infections.

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Interferon increases the amount of USP18.

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Interferon increases the amount of USP18. 7 / 9

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Like ISG15, the expression of its conjugating enzymes and USP18 is upregulated by IFN (Fig. 1a) .

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Previous studies have shown that the expression of USP18 could be rapidly induced by interferon, viral infection, and genotoxic stress XREF_BIBR.

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ISG43 expression is induced by interferon and negatively regulated by RNase-L.

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The expression of Mx2, Rsad2, Ifit3, Herc5 and Usp18, but not RnaseL and Ifitm3, was significantly induced by the IFN injection in the PBS control group.

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Like ISG15, the expression of its conjugating enzymes and USP18 is upregulated by IFN (XREF_FIG a).

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To investigate whether endogenous IFN signaling induces USP18 expression during tumor development in vivo, we inoculated C57BL/6 mice with B16-GFP tumor cells.

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However, USP18 expression is strongly stimulated by IFN treatment and exerts negative regulation of type I interferon signaling, which is independent of its enzymatic activity 21.

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Interferon inhibits USP18.

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Interferon inhibits USP18. 2 / 2

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As shown in Fig. 4 B, anti-IFN alpha/beta antibodies significantly reduced expression of the interferon stimulated genes ISG15 and USP18.

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The decrease was associated with the expression of IFN stimulated gene USP18 (UBP43), which is proposed to interact with the IFN-alpha receptor and inhibit signaling through JAK1.

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USP18 affects IFNA

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USP18 inhibits IFNA.

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USP18 inhibits IFNA. 10 / 26

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We speculate that by blocking IFN-alpha signaling, USP18 expression may lead to an enhanced susceptibility to infection with interferon sensitive viruses and enhanced viral proliferation.

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IFN-lambda4 potently blocks IFN-alpha signalling by ISG15 and USP18 in hepatitis C virus infection .

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Lack of USP18 was shown to abrogate this desensitizing effect of IFN-alpha in vivo [XREF_BIBR], whereas USP18 expression was sufficient to establish this state of refractoriness [XREF_BIBR].

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The reason for this paradox is unknown , but IFN-lambda4 has been speculated to render HCV-infected hepatocytes refractory to IFN-alpha , for example by inducing the expression of USP18 , which inhibits IFN-alpha but not IFN-lambda signaling41 .

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The results suggested that USP18 silencing augments the antiviral effects of IFN-alpha against HBV expression (XREF_FIG).

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Studies from ours and others demonstrated that higher expression levels of USP18 inhibited IFN-a anti-HBV and HCV activity in chronic HBV- and HCV infected patients [XREF_BIBR, XREF_BIBR].

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The reason for this paradox is unknown, but IFN-λ4 has been speculated to render HCV-infected hepatocytes refractory to IFN-α, for example by inducing the expression of USP18, which inhibits IFN-α but not IFN-λ signaling41.

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USP18 silencing enhances the antiviral activity of IFN-alpha through JAK-STAT signaling pathway.

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Of note, USP18 knockdown did not increase pSTAT1 in response to IFN-alpha before 24 h; these findings are consistent with previous observations.

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The reason for this paradox is unknown, but IFN-lambda4 has been speculated to render HCV infected hepatocytes refractory to IFN-alpha, for example by inducing the expression of USP18, which inhibits IFN-alpha but not IFN-lambda signaling XREF_BIBR.

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USP18 activates IFNA.

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USP18 activates IFNA. 10 / 20

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The IFN-alpha signal blocking activity of USP18 is mediated by the binding of USP18 to the intracellular domain of IFNAR2, which prevents the binding of JAK1 to IFNAR2 27.

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In addition, lacking USP18 can abrogated the refractory status induced by IFN-alpha stimulation, which indicates that lack of USP18 in the Hepg2.2.15 cells could enhance the antiviral activity of IFN-alpha (XREF_SUPPLEMENTARY).

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In the current study, for the first time, we demonstrated that ISG15 and USP18 protein levels are increased in HCV infected PHHs and IFN-lambda4-expressing or -treated cells and that ISG15 and USP18 proteins mediate IFN-alpha unresponsiveness in IFN-lambda4-expressing or -treated cells.

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Taken together, we concluded that USP18 potentiates the anti-HBV activity of IFN-alpha by targeting the JAK and STAT signaling pathway in Hepg2.2.15 cells.

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Namely, free extracellular ISG15 is crucial in IFN-gamma-dependent antimycobacterial immunity, while free intracellular ISG15 is crucial for USP18 mediated downregulation of IFN-alpha and beta signalling.

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Furthermore, previous studies reported that blocking of IFN-α signaling by USP18 does not depend on the enzymatic activity of USP1827.

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The ISG15 and USP18 mediated IFN-alpha unresponsiveness was demonstrated by transfection of siRNAs targeting ISG15 and/or USP18.

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Usp18 deficient cells have enhanced IFN-alpha and beta signaling and more ISG15 modified proteins.

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The ISG15/USP18-mediated IFN-α unresponsiveness was demonstrated by transfection of siRNAs targeting ISG15 and/or USP18.

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Our results indicated that USP18 modulates the anti-HBV activity of IFN-alpha via activation of the JAK and STAT signaling pathway in Hepg2.2.15 cells.

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USP18 affects apoptotic process

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USP18 inhibits apoptotic process.

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USP18 promotes cell proliferation and suppressed apoptosis in cervical cancer cells via activating AKT signaling pathway.

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Subsequently, we demonstrated that knockdown of USP18 inhibited HCC cell growth and caused cell-cycle arrest and early apoptosis.

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83 The absence of USP18 in the breast cancer cell line MCF-7 results in an increase in the induction of apoptosis by chemotherapy and treatment with IFN-α.27 Silencing USP18 in glioblastoma cells produces similar results,84 a finding suggesting that strengthening the IFN-I pathway by silencing USP18 elicits apoptosis in drug-treated cells with robust caspase-8 and caspase-3 activation independent of the mitochondrial pathway.27The direct effect of USP18 on tumor progression has been studied with leukocytes.

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Overall, these results suggested that USP18 promotes pancreatic cancer cell proliferation by facilitating cell cycle progression and inhibiting cell apoptosis.

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Silencing of USP18 in hepatoma HepG2 cells was shown to cause accumulation of cells in G0/G1 and apoptosis 29.

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Deubiquitinase Usp18 prevents cellular apoptosis from oxidative stress in liver cells.

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In fact, double inhibition of USP18 and STAT1 or STAT2 reverted the supportive effect of USP18 knockdown in IFNalpha induced beta cell apoptosis.

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Subsequent work showed that knockdown of USP18 destabilized PML-RARalpha and promoted apoptosis in NB4 APL cells but did not have an effect on differentiation (Guo et al., 2010).

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Silencing USP18 in SiHa and Caski cervical cancer cell lines inhibited cell proliferation, induced apoptosis, and promoted cleaved caspase-3 expression.

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Besides, USP18 silencing promoted the early apoptosis of HCC cells.

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USP18 activates apoptotic process.

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Indeed, it has been shown that ablating USP18, the enzyme that deconjugates ISG15 from target proteins, increased TRAIL production and promoted the extrinsic apoptosis pathway in cells treated with IF[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Also, depletion of Usp18 led to a decrease in protein levels of other known oncogenic targets of miR-7, reduced cell proliferation and soft agar colony formation, and increased apoptosis.

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Downregulation of USP18 inhibits growth and induces apoptosis in hepatitis B virus related hepatocellular carcinoma cells by suppressing BCL2L1.

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Consistently, the loss of c-Myc reversed the cell cycle arrest and apoptosis induced by USP18 overexpression in SW1990 cells (XREF_FIG - XREF_FIG).

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Therefore, all the above studies demonstrated that ISG15 and USP18 alone induced apoptosis in leukemia, myeloma and cervical cancer cells.

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XREF_BIBR - XREF_BIBR Consistent with this, an siRNA to the ISG, USP18, that deconjugates ISG15 from target proteins, increased expression of TRAIL and thus promoted apoptosis.

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27 Silencing USP18 in glioblastoma cells produces similar results, 84 a finding suggesting that strengthening the IFN-I pathway by silencing USP18 elicits apoptosis in drug treated cells with robust caspase-8 and caspase-3 activation independent of the mitochondrial pathway.

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Down-regulation of USP18 expression together with the induction of ER-stress efficiently restored apoptosis in U87MG cells.

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More importantly, ISG15 and USP18 induced cancer cell apoptosis.

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Identification of USP18 as an important regulator of the susceptibility to IFN-alpha and drug induced apoptosis.

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FIG 5 : IFN-alpha , LPS , and TNF-alpha do not induce type 1/2 IFN in primary murine hepatocytes but do induce USP18 .

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USP18 is induced in hepatocytes by LPS and TNF - but not by IL-6 and IL-10 .

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Usp18 is strongly upregulated by type I and type III IFNs [ 33,35,37,51,52 ] , lipopolysaccharides [ 53,54 ] , polyI :C [ 53 ] , tumor necrosis factor alpha ( TNFalpha ) [ 54 ] , or genotoxic stresses [ 35,55 ] .

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For example, inflammatory stimuli, e.g., endotoxin and lipopolysaccharide (LPS), have been shown to upregulate USP18 in peritoneal exudate macrophages (18).

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We next sought to determine whether TNF-alpha and LPS could induce USP18 expression in hepatocytes via the induction of IFN-alpha .

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For example, inflammatory stimuli, e.g., endotoxin and lipopolysaccharide (LPS), have been shown to upregulate USP18 in peritoneal exudate macrophages.

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For example, LPS treatment of a murine macrophage cell line upregulates USP18 in an IRF3 dependent manner.

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Bone marrow derived macrophages were isolated from wild type (WT), USP18 MKO or TAK1 MKO mice and treated with LPS or CpG, the expressions of cytokines including IL-6, IL-10, IL-1beta, and TNF-alpha were measured.

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FIG 1 : USP18 expression is upregulated by TNF-alpha and LPS .

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USP18 can also be induced by lipopolysaccharide ( LPS ) stimulation or virus infection ( Li et al ., 2016 ; MacParland et al ., 2016 ) .

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No evidence text available

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We next sought to determine whether TNF-alpha and LPS could induce USP18 expression in hepatocytes via the induction of IFN-alpha.

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TNF-alpha and LPS treatment also led to augmented USP18 protein expression by Western blotting (XREF_FIG) and by intracellular staining (XREF_FIG to XREF_FIG).

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We observed that neither IFN-α, LPS, nor TNF-α induce much, if any, hepatocyte expression of IFN-α or IFN-γ, although the same doses induce strong expression of USP18 (Fig. 5).

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USP18 mRNA expression was induced by TNF-α and LPS but not by IL-6 or IL-10 (Fig. 1A).

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MacParland et al. showed that TNF-alpha or LPS could target USP18 expression and thus inhibit IFN signaling [XREF_BIBR].

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TABLE 1: Inhibition of inflammatory signaling impairs TNF-α- and LPS-induced USP18 expressionaFIG 1: USP18 expression is upregulated by TNF-α and LPS.

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LPS strongly activates UBP43 expression in macrophages, which is paralleled by changes in UBP43 protein levels.

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USP18 mRNA expression was induced by TNF-alpha and LPS but not by IL-6 or IL-10 (XREF_FIG).

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We next sought to determine whether TNF-α and LPS could induce USP18 expression in hepatocytes via the induction of IFN-α.

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As seen in Fig. 6B, HIRI led to a significant increase in LCMV viral titers in USP18+/+ mice, an effect that was not observed in USP18−/− mice.Having shown that LPS/TNF-α stimulation increases hepatocyte USP18 expression, we then sought to determine whether we could pharmacologically inhibit the induction of USP18 by LPS and TNF-α.

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Having shown that LPS and TNF-alpha stimulation increases hepatocyte USP18 expression, we then sought to determine whether we could pharmacologically inhibit the induction of USP18 by LPS and TNF-alpha.

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Inhibition of inflammatory signaling impairs LPS and TNF-alpha stimulated USP18 induction.

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Having shown that LPS / TNF - stimulation increases hepatocyte USP18 expression , we then sought to determine whether we could pharmacologically inhibit the induction of USP18 by LPS and TNF - .

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Having shown that LPS / TNF-alpha stimulation increases hepatocyte USP18 expression , we then sought to determine whether we could pharmacologically inhibit the induction of USP18 by LPS and TNF-alpha .

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This is consistent with our finding that inhibiting Ifit1 using siRNA suppressed Usp18 and Mx1 expression in response to LPS.

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ISG15 affects USP18

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ISG15 activates USP18.

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Indeed, individuals lacking ISG15 expressed lower levels of ubl carboxy-terminal hydrolase 18 (uSP18), which was rescued by complementation with either wild-type or a non-conjugatable form of ISG15 (ref.

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Decreasing ISGylation by knockdown of the ISG15 E1 enzyme, Ube1L, in primary USP18(+/+) and USP18(−/−) hepatocytes led to increased MHV-3 replication.

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Moreover, we confirm that ISG15 promotes USP18 accumulation independently of conjugation 8.

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ISG15 is known to promote USP18-mediated inhibition of type I 437 IFN signaling by stabilizing USP18 activity and preventing its proteasomal degradation(Zhang et 438 al., 2015), underscoring the role of ISG15 as a negative regulator of type I IFN responses when 439 co-upregulated with USP18.

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USP18 exerts a negative regulatory effect on type I interferon signalling by competing with JAK1 for IFNAR2 binding, and mutations in USP18 and ISG15, which directly regulates USP18 stability, result in aberrant type I interferon induction in humans .

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Recently, we found that prolonged exposure to IFN- up-regulates U-ISGF3 and U-ISGs, including ISG15, and that ISG15 causes the refractoriness to exogenous IFN- treatment by stabilizing USP18 protein [60] .

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ISG15 prevents the degradation of USP18 by sphingosine kinase 2 (SPK2) [XREF_BIBR, XREF_BIBR].

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9 This phenotype cannot be explained by the delSGylation activity of USP18, because deleting ISG15 or the ISGylation-activating Functions of USP18 N Honke et al enzyme UBE1L in mice did not reverse the phenotype in Usp18-deficient mice.

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Recently, we found that prolonged exposure to IFN-λ up-regulates U-ISGF3 and U-ISGs, including ISG15, and that ISG15 causes the refractoriness to exogenous IFN-α treatment by stabilizing USP18 protein [60].

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ISG15 inhibits USP18.

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This lack of intracellular free ISG15 prevents the accumulation of USP18, a known negative regulator of IFN-alpha and beta, resulting in enhanced IFN-alpha and beta immunity and autoinflammation, resembling Aicardi-Goutieres syndrome and spondyloenchondromatosis [XREF_BIBR, XREF_BIBR, XREF_BIBR].

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In a follow-up report, Zhang et al. show that the absence of intracellular ISG15 in patients ' cells also prevents the accumulation of USP18, a potent negative regulator of type I IFN [XREF_BIBR].

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We therefore analysed the impact of ISG15 on SKP2 mediated USP18 degradation.

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The authors also showed that intracellular ISG15 deficiency prevented ubiquitin specific peptidase 18 (USP18) accumulation.

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Decreasing ISGylation by knockdown of the ISG15 E1 enzyme, Ube1L, in primary USP18(+/+) and USP18(−/−) hepatocytes led to increased MHV-3 replication.

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We further show that an absence of intracellular ISG15 in the patients ' cells prevents the accumulation of USP18 XREF_BIBR, XREF_BIBR, a potent negative regulator of IFN-alpha and beta signalling, resulting in the enhancement and amplification of IFN-alpha and beta responses.

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Recently, human ISG15 deficiency was found to cause a decrease in USP18 accumulation and this was hypothesized to cause the loss of negative feedback of type I interferon signaling in these patients leading to auto-inflammation [XREF_BIBR].

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| 2

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As expected from the previous study 10, silencing ISG15 expression decreased the protein level of USP18 in IFN-lambda4-transfected cells.

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As expected from the previous study10, silencing ISG15 expression decreased the protein level of USP18 in IFN-λ4-transfected cells (Fig. 5C).

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ISG15 increases the amount of USP18.

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ISG15 silencing decreased the amount of USP18 protein in recombinant IFN-lambda4-treated cells, and the protein level of USP18 was restored not only by transfection of wild type (WT) ISG15 gene but also by transfection of conjugation defective ISG15 AA mutant gene.

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ISG15 silencing decreased the amount of USP18 protein in recombinant IFN-λ4-treated cells, and the protein level of USP18 was restored not only by transfection of wild type (WT) ISG15 gene but also by transfection of conjugation-defective ISG15 AA mutant gene (Supplementary Fig. 8A).

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As expected from the previous study10, silencing ISG15 expression decreased the protein level of USP18 in IFN-λ4-transfected cells (Fig. 5C).

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ISG15 deubiquitinates USP18.

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The coexpression of either wild-type ISG15 or ISG15 (DeltaGG) with USP18 and ubiquitin resulted in markedly lower levels of USP18 ubiquitination (XREF_FIG, lanes 9-11 and XREF_FIG) and larger total amounts of USP18.Overall, these data indicate that free intracellular ISG15 antagonizes USP18 ubiquitination and degradation, thereby promoting the stability and function of this protein.

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The non-covalent interactions of ISG15 and USP18 prevent the ubiquitination of USP18 by S-phase kinase-associated protein two and stabilize the downregulation of the IFN signaling pathway by USP18 (Tokarz et al., 2004; Zhang et al., 2015).

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TNF increases the amount of USP18.

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We suspect that whereas increased TNF-α does contribute to USP18 expression, there are other stimuli, for example, LPS and perhaps the recently described interferon-lambda 4 (56), that also modulate hepatic USP18 expression.

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USP18 mRNA expression was induced by TNF-alpha and LPS but not by IL-6 or IL-10 (XREF_FIG).

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We suspect that whereas increased TNF-alpha does contribute to USP18 expression, there are other stimuli, for example, LPS and perhaps the recently described interferon-lambda 4, that also modulate hepatic USP18 expression.

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As seen in Fig. 7A and as described in Table 1, TNF-α induced expression of USP18 was potently downregulated by all inhibitors tested, and this inhibition coincided with impaired IL-1β mRNA expression (Fig. 7B).

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Treatment of Huh7.5 cells with TNF-α or LPS induced expression of USP18 in 74.1% ± 8.9% and 70.5% ± 5.2%, respectively, compared to untreated controls in which USP18 expression was 31.0% ± 4.7% (Fig. 1Fi).

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We next sought to determine whether TNF-α and LPS could induce USP18 expression in hepatocytes via the induction of IFN-α.

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TABLE 1: Inhibition of inflammatory signaling impairs TNF-α- and LPS-induced USP18 expressionaFIG 1: USP18 expression is upregulated by TNF-α and LPS.

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We observed that neither IFN-α, LPS, nor TNF-α induce much, if any, hepatocyte expression of IFN-α or IFN-γ, although the same doses induce strong expression of USP18 (Fig. 5).

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We next sought to determine whether TNF-alpha and LPS could induce USP18 expression in hepatocytes via the induction of IFN-alpha.

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These data suggest that the induction of USP18 by TNF - and LPS , and pos-sibly other inflammatory stimuli , is promoted by NF - > signaling and that hepatocyte USP18 expression in particular-compared to IL-1 - may be an attractive target for pharmacologic manipulation in the setting of liver inflammation .

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USP18 is induced in hepatocytes by LPS and TNF-alpha but not by IL-6 and IL-10.

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As seen in Fig. 7A and as described in Table 1 , TNF-alpha induced expression of USP18 was potently downregulated by all inhibitors tested , and this inhibition coincided with impaired IL-1beta mRNA expression ( Fig. 7B ) .

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These data suggest that the induction of USP18 by TNF-α and LPS, and possibly other inflammatory stimuli, is promoted by NF-κΒ signaling and that hepatocyte USP18 expression in particular—compared to IL-1β—may be an attractive target for pharmacologic manipulation in the setting of liver inflammation.In this study we examined the role of various inflammatory stimuli in the induction of USP18 and the downstream establishment of an IFN-α refractory state.

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FIG 1 : USP18 expression is upregulated by TNF-alpha and LPS .

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We suspect that whereas increased TNF - does contribute to USP18 expression , there are other stimuli , for example , LPS and perhaps the recently described interferon-lambda 4 ( 56 ) , that also modulate hepatic USP18 expression .

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USP18 expression is upregulated by TNF - and LPS .

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These data suggest that the induction of USP18 by TNF-alpha and LPS, and possibly other inflammatory stimuli, is promoted by NF-kappaBeta signaling and that hepatocyte USP18 expression in particular -- compared to IL-1beta -- may be an attractive target for pharmacologic manipulation in the setting of liver inflammation.

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FIG 5 : IFN-alpha , LPS , and TNF-alpha do not induce type 1/2 IFN in primary murine hepatocytes but do induce USP18 .

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19 The Usp18 gene is rapidly and strongly upregulated after viral infection or by type I and type III IFNs, 1, 6, 13, 19, 20 lipopolysaccharide (LPS), 21, 22 tumor necrosis factor alpha (TNF-α), 22 or genotoxic stress 13, 23 (Figure 2a ).

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Inhibition of inflammatory signaling impairs LPS and TNF-alpha stimulated USP18 induction.

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USP18 affects NFkappaB

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USP18 inhibits NFkappaB.

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The LPS-mediated TLR4 activation in human macrophages upregulates USP18 , which in turn inhibits NF-kappaB activation , and thus the secretion of pro-inflammatory cytokines ( TNF-alpha , IL-6 , and IL-1beta ) via interacting with TAK1-TAB1 and IKKalpha / beta-NEMO complexes ( Table 2 ) [ 193 ] .

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The LPS mediated TLR4 activation in human macrophages upregulates USP18, which in turn inhibits NF-kappaB activation, and thus the secretion of proinflammatory cytokines (TNF-alpha, IL-6, and IL-1beta) via interacting with TAK1-TAB1 and IKKalpha and beta-NEMO complexes [193].

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USP18 was upregulated by various TLR ligands in THP-1 (a human monocyte cell line) cells and inhibited IkappaB degradation as well as NF-kappaB activation to form a negative feedback loop.

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Another way that USP18 inhibited NF-kappaB activation is by deubiquitinating K63-Ub of TAK1 and NEMO [ 42 ] .

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Here, we found that ectopic expression of USP18 suppressed nuclear accumulation of p65 as well as NF-kappaB activation by LPS treatment.

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USP18 deficiency or knockdown of USP20 resulted in enhanced K48 linked ubiquitination and accelerated degradation of STING, and impaired activation of IRF3 and NF-kappaB as well as induction of downstream genes after infection with DNA virus HSV-1 or transfection of various DNA ligands.

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USP18 inhibits NF-kappaB signaling at the level of the IKK complex.

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Another way that USP18 inhibited NF-kappaB activation is by deubiquitinating K63-Ub of TAK1 and NEMO [XREF_BIBR].

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A recent study showed that USP18 attenuated NF-kappaB activation by targeting TAK1-TAB1 for deubiquitination.

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XREF_BIBR This study showed that USP18 or USP20 deficiency significantly inhibited HSV-I or cytosolic DNA activation of both NF-kappaB and IRF3, and type-I IFN and proinflammatory cytokine expression.

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Knockdown of USP18 enhances NF-kappaB activation as well as the inflammatory response.

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These results suggested that the suppression of NF-kappaB signaling by USP18 is mainly dependent on the inhibition of K63 linked polyubiquitination of NEMO on lysine residues at positions 325 and 326.

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Therefore it is tempting to suggest that early induction of NF-kappaB (24hpi) mediated by USP18 during PRRSV infection, via increasing and decreasing nuclear translocation of p65 and p50, respectively, is detrimental for viral growth.

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USP18 affects ISG15

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Immunoprecipitation (IP) assays confirmed that ISG15 directly complexed with PTEN protein and this conjugation was attenuated by engineered overexpression of USP18.

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In particular, replacement of the Ala138 in USP18 by a polar residue in other USPs might block the access of the bulky and hydrophobic Trp121 side chain of ISG15 (XREF_FIG).

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As expected, USP18 silencing dramatically decreased USP18 protein and increased the free ISG15 protein compared to the control group (XREF_FIG).

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Here, a clear negative feedback loop can be observed, in which Ubiquitin like protein ISG15 (G1P2) induces IFN-gamma expression while Ubl carboxyl-terminal hydrolase 18 (USP18) inhibits G1P2 activity, explaining the observed down-regulation state of IFN-gamma.

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In particular, replacement of the Ala138 in USP18 by a polar residue in other USPs might block the access of the bulky and hydrophobic Trp121 side chain of ISG15 (Fig. 4d).

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ISG15 conjugation (ISGylation) can be reversed by the IFN-a/b-induced isopeptidase (USP18) that cleaves ISG15 from target proteins 4 .

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USP18 activity reduces ISG15 protein conjugation and promotes tumorigenesis if an oncogenic substrate is stabilized by USP18.

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Inhibiting USP18, the negative regulator of ISGylation, has been shown in both cell line and invivo systems to enhance ISG15 conjugation (Ketscher et al., 2015).

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Finally, USP18 (an ISG15 specific isopeptidase enzyme) can reverse ISG15 conjugation of target proteins.

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In a positive feedback loop, the ISG15 conjugation system is also upregulated by p53 ISGylation to further potentiate p53 transactivity and downregulated by USP18 mediated deISGylation of p53.

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However divergent, ISG15 removal is nevertheless exclusively mediated by USP18 in humans, mice, and zebrafish.

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In addition, absence of USP18, which cleaves ISG15 from its target protein, prolongs ISG15 mediated ISGylation.

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USP18 can increase HBV susceptibility by removing isopeptidase activity of ISG15 and promoting HBV replication by downregulating the I-IFN signal transduction pathway.

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At later stages, USP18 mediated de-ISGylation releases free ISG15, which in turn stabilizes USP18 and tunes down IFN-alpha and beta signalling and inflammation.

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The absence of USP18 strengthens the signaling of IFN-I and IFN-III; 1,2 and is associated with prolonged Janusactivated kinase/signal transducer and activator of In addition, absence of USP18, which cleaves ISG15 from its target protein, prolongs ISG15-mediated ISGylation.

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Interestingly, this enhanced type I interferon signalling has not been observed in mice (Box 1), raising interesting questions about the divergent function of ISG15 between species.The regulation of interferon signalling by ISG15 and USP18 also contributes to the unexpected proviral activity for ISG15 that has been associated with chronic viral hepatitis.

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Our data are consistent with this mechanism, since the blunting of hepatocyte IFN signaling after exposure to inflammatory stimuli is independent of USP18 mediated removal of ISG15 from its target proteins.

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Usp18 deficient cells have enhanced IFN-alpha and beta signaling and more ISG15 modified proteins.

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Unlike Lbpro, USP18-mediated ISG15 cleavage leads to ISG15 recycling since USP18 cleaves the isopeptide linkage after the C-terminal GlyGly motif and ISG15 remains competent for reconjugation.

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We demonstrated that USP18 deficient cells have increased expression of IFIT1, IFIT2, IFIT3, IFITM1, IFITM2, CXCL9, CXCL10, ISG15, and MX2 after treatment with exogenous IFN-. Many of these genes have been previously shown to restrict HIV-1 replication.

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We demonstrated that USP18 deficient cells have increased expression of IFIT1, IFIT2, IFIT3, IFITM1, IFITM2, CXCL9, CXCL10, ISG15, and MX2 after treatment with exogenous IFN‐β.

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IFNA activates USP18.

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Interestingly, unlike the results shown in XREF_FIG for ISG15, ISG56 and ISG12, we observed little or no IFN-alpha induction of the ISG43 mRNA by northern blotting in A. 2 Akata cells (which exhibited high and prolonged ISG expression), whereas induction of ISG43 expression was easily detectable in A. 15 cells (data not shown).

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Supporting this idea , we could verify that USP18 was expressed in both Tregs and Tconvs and was strongly induced by IFNa in a time-and dose-dependent fashion , as measured by intracellular staining of the USP18 protein ( not shown ) .

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The defect in the UBP43-mediated negative feedback control of IFN signaling that we have uncovered is in the IFN-α-induced expression of UBP43, either in the transcriptional activation of the UBP43 gene by IFN-α, or in a co- or post-transcriptional event that results in a specific reduction in UBP43 mRNA production.

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In our microarray analysis of IFN treated PHH, USP18 was induced preferentially by IFN-alpha.

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FIG 5 : IFN-alpha , LPS , and TNF-alpha do not induce type 1/2 IFN in primary murine hepatocytes but do induce USP18 .

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Indeed, USP18 was induced almost exclusively by IFN-alpha.

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Furthermore, knockdown of USP18 increases both ISG induction and anti-HCV activity of IFN-alpha, and data have shown that IFN-alpha treatment given to mice in vivo increases hepatic USP18 and blunts the effect of a subsequent dose of IFN-alpha.

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Furthermore, knockdown of USP18 increases both ISG induction and anti-HCV activity of IFN-α (14), and data have shown that IFN-α treatment given to mice in vivo increases hepatic USP18 and blunts the effect of a subsequent dose of IFN-α (12).

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Supporting this idea , we could verify that USP18 was expressed in both Tregs and Tconvs and was strongly induced by IFNalpha in a time - and dose-dependent fashion , as measured by intracellular staining of the USP18 protein ( not shown ) .

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In addition, we show that IFN-alpha upregulates ISG15 and USP18 via STAT1, and that, in turn, each of these 2 proteins alone can mimic the effects of IFN-alpha on neurogenesis.

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We observed that neither IFN-α, LPS, nor TNF-α induce much, if any, hepatocyte expression of IFN-α or IFN-γ, although the same doses induce strong expression of USP18 (Fig. 5).

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In contrast to IFN-l treatment, IFN-a treatment of this clone failed to induce the expression of the IFN-stimulated genes, Oasl2 ( Fig. 2A) and Usp18 (Fig. 2B) , and to protect against infection with TM967, a TMEV derivative expressing mCherry (Fig. 2C) .

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Untreated INS-1E cells showed low USP18 mRNA expression, but IFNalpha upregulated USP18 expression from 2 to 24h (XREF_FIG).

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IFN-alpha, LPS, and TNF-alpha do not induce type 1 and 2 IFNs in primary murine hepatocytes but do induce USP18 expression.

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Thus, the induction of ISGs by LPS and TNF-α is very unlikely to reflect the induction of hepatocyte type 1 or type 2 IFN, which suggests that the observed USP18 induction is not due to type 1 or type 2 IFN secretion and autocrine stimulation of the IFN-α receptor.We then assessed whether tissue-wide hepatic inflammatory stress increases liver USP18 expression, as an in vivo confirmation of our in vitro findings.

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XREF_BIBR, XREF_BIBR We presently show that IFNalpha and dsRNA induce USP18 expression in human islets, primary rat beta cells and INS-1E cells, supporting its role in IFN related signaling pathways and antiviral responses in pancreatic beta cells.

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We indeed demonstrated that both concentrations of IFN-alpha (500 and 5000 pg/mL) significantly increased mRNA gene expression of STAT1 (fold change [fc] = 2.7 and 3.1, respectively), ISG15 (fc = 2.1 and 3.3), and USP18 (fc = 2.1 and 3.4) (XREF_FIG).

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Table: IFNA treatment

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In contrast to IFN-λ treatment, IFN-α treatment of this clone failed to induce the expression of the IFN-stimulated genes, Oasl2 (Fig. 2A) and Usp18 (Fig. 2B), and to protect against infection with TM967, a TMEV derivative expressing mCherry (Fig. 2C).

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Table 3. Genes on chromosome 22 exhibiting IFN-sensitive expression changes

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IFNA inhibits USP18.

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IFNA inhibits USP18. 3 / 3

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The IFN-alpha signal blocking activity of USP18 is mediated by the binding of USP18 to the intracellular domain of IFNAR2, which prevents the binding of JAK1 to IFNAR2 27.

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The IFN-α signal-blocking activity of USP18 is mediated by the binding of USP18 to the intracellular domain of IFNAR2, which prevents the binding of JAK1 to IFNAR2 27 .

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The IFN-α signal-blocking activity of USP18 is mediated by the binding of USP18 to the intracellular domain of IFNAR2, which prevents the binding of JAK1 to IFNAR227.Our group recently found that prolonged stimulation of IFN-λ3 induces the robust and sustained upregulation of ISG15 that stabilizes the USP18 protein and causes the unresponsiveness to exogenous IFN-α treatment10.

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USP18 affects cell population proliferation

| 1 19

USP18 activates cell population proliferation.

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USP18 activates cell population proliferation. 10 / 18

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In present study, our data indicated that knockdown of USP18 could inhibit cell proliferation and induce cell cycle arrest in the G1 phase in HCC cells.

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Furthermore, knockdown of USP18 significantly suppressed tumour cell proliferation in vitro and tumour growth in vivo, whereas overexpression of USP18 promoted HCC cells growth.

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This study showed that USP18 can promote the proliferation of colorectal cancer cells and might be a potential biomarker for the diagnosis of CRC.

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USP18 positively regulates cell proliferation.

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Simultaneously, CCK8 and EdU assays showed that the reduced proliferation induced by USP18 knockdown in BxPC-3 cells was partly abolished by the introduction of c-Myc (XREF_FIG - XREF_FIG).

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However, USP18 silencing blocked the proliferation and migration induced by FTO.

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Wound healing assays suggested that USP18 promoted the proliferation of CRC cells.

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We further demonstrated that overexpression of USP18 modestly enhances proliferation of kidney cells and Usp18 is overexpressed in a mouse model of Wilms tumor.

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This suggests that USP18 positively modulates cell proliferation.

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USP18 promotes cell proliferation and suppressed apoptosis in cervical cancer cells via activating AKT signaling pathway.

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USP18 inhibits cell population proliferation.

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USP18 inhibits cell population proliferation. 4 / 4

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Further studies demonstrated that ectopic ISG15 and USP18 inhibited proliferation of myeloma, leukemia and cervical cancer cells.

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Further studies demonstrated that ectopic ISG15 and USP18 inhibited proliferation of myeloma, leukemia and cervical cancer cells.

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Ectopic overexpression and knockdown assays indicated that USP18 can negatively regulate the proliferation of PTC cell lines.

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As shown in Figure XREF_FIG, ISG15 and USP18 individually suppressed HeLa cell proliferation.

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USP18 affects STAT

| 12

USP18 inhibits STAT.

| 10

USP18 inhibits STAT. 10 / 13

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Suppression of USP18 activated the JAK and STAT signaling pathway as shown by the increased and prolonged expression of phosphorylated signal transducer and activator of transcription 1 (p-STAT1) in combination with enhanced expression of several interferon stimulated genes (ISGs).

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As expected, exogenous USP18 expression significantly inhibited IFN-a-induced Jak and STAT signaling as shown by decreased p-STAT1 levels and ISRE activity.

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Protein lysates were collected on day 4 postinfection and Gagp24 and USP18 expression were assessed by Western blotFigure 4: siRNA knockdown of USP18 enhances STAT activation and expression of ISGs in IFN‐β‐treated MDMs.

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After 24 h, genomic DNA was collected and a qPCR assay developed to measure integrated proviral genomes showed that there were on average 3800 genomes per 10,000 cells in USP18 +/+ iMacs and on average 4900F I G U R E 4 siRNA knockdown of USP18 enhances STAT activation and expression of ISGs in IFN--treated MDMs.MDMs from 6 donors were transfected with nontargeting (NT) control or USP18 siRNA for 3 h followed by IFN-treatment for 18 h. (A) Expression of p-STAT1, p-STAT2, and USP18 was measured by Western blot.

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It has been reported that C-terminal region of USP18 (amino acid residue 312-368) competes with Jak1 for interacting with the type I IFN receptor (IFNAR2) and represses downstream Jak and STAT signaling pathway [XREF_BIBR].

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Under physiological conditions, USP18 inhibits STAT signaling, decreasing IFNalpha induced chemokine production and activation of several members of the BH3-only protein family.

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We next examined the effect of USP18 inhibition on the kinetics of IFNalpha induced STAT signaling activation.

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Our findings indicate that USP18 inhibition induces inflammation by increasing the STAT signaling and exacerbates IFN induced beta cell apoptosis by the mitochondrial pathway of cell death.

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Taken together, we concluded that USP18 potentiates the anti-HBV activity of IFN-alpha by targeting the JAK and STAT signaling pathway in Hepg2.2.15 cells.

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Moreover, USP18 competitively inhibits IFN-alpha and beta-induced JAK and STAT activation [XREF_BIBR] and upregulates epidermal growth factor receptor (EGFR) expression [XREF_BIBR].

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USP18 activates STAT.

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USP18 activates STAT. 2 / 5

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In contrast, silencing USP18 activates the Jak and STAT signaling and potentiates IFN anti-HCV ativity [XREF_BIBR].

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Ye et al. (2021) demonstrated that DENV-2 infection increased USP18 expression; USP18 overexpression enhanced DENV-2 replication, while USP18 silencing inhibited DENV-2 replication by activating the IFN-α-mediated JAK/STAT signaling pathway.

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USP18 affects EGFR

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USP18 activates EGFR.

| 9

USP18 activates EGFR. 9 / 10

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In addition, the results indicated that USP18 may promote the epidermal growth factor (EGF)-mediated EGF receptor (EGFR)/AKT/S-phase kinase associated protein2 (Skp2) pathway by upregulating EGFR and Skp2 in a AKT and forkhead boxO3 dependent manner in breast cancer.

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However, USP18 has independent direct effects on tumor growth and survival and enhances EGFR signaling by decreasing miR-7 expression, which targets EGFR XREF_BIBR.

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For instance, Tan et al. found that USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway [XREF_BIBR].

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USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway.

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Moreover, USP18 competitively inhibits IFN-alpha and beta-induced JAK and STAT activation [XREF_BIBR] and upregulates epidermal growth factor receptor (EGFR) expression [XREF_BIBR].

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Inhibition of USP18 reduces the levels of EGFR and other oncogenic proteins and inhibits the tumorigenic activity of cancer cells.

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Tan et al. have demonstrated that USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway.

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However, USP18 has independent direct effects on tumor growth and survival and enhances EGFR signaling by decreasing miR-7 expression, which targets EGFR [54].

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USP18 increases the amount of EGFR.

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USP18 increases the amount of EGFR. 2 / 2

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In an RNA interference screen of 106 different genes related to deubiquitination, silencing of USP18 reduced EGFR expression without affecting the levels of other receptor tyrosine kinases [XREF_BIBR].

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USP18 inhibits SGCG. 8 / 8

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While we understand well how USP18 recognises ISG15 and deconjugates it from target proteins, it is less clear how USP18 negatively regulates type I IFN signalling on the molecular level.

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Recent data by Zhang and coworkers (Malakhova et al., 2006) revealed, however, that USP18 attenuates JAK-STAT signaling, and thereby the type 1 IFN response, in a non-enzymatic manner, i.e., by directly competing with JAK1 for binding to the IFNAR2 subunit of the type 1 IFN receptor.

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Indeed, IRF1 uniquely promotes the transcription of the negative regulator USP18 (Forero et al., 2019), which selectively inhibits type I IFN signaling without affecting IFN-λ signaling (Blumer et al., 2017).

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Besides being an active enzyme, USP18 negatively regulates type I interferon signalling independent of its protease activity [22] (Figure 1).

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USP18 also interacts with IFNAR2 and STAT2 to block type I interferon signalling in a protease-independent manner.

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In addition to its isopeptidase activity, USP18 negatively regulates type I and type III IFN signalling by blocking the IFNAR2 subunit of the interferon receptor 184 .

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CRISPR/Cas9 knockout of USP18 enhances type I IFN responsiveness and restricts HIV-1 infection in macrophages.The IFN-stimulated gene ubiquitin-specific proteinase 18 (USP18) encodes a protein that negatively regulates T1 IFN signaling via stearic inhibition of JAK1 recruitment to the IFN-receptor 2 subunit (IFNAR2).

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Different infection models for hepatitis C and B virus (HCV and HBV) revealed that silencing of USP18 in hepatoma cell lines potentiates the antiviral activity of type I interferons against HBV and HCV [9,47].

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USP18 activates SGCG.

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USP18 activates SGCG. 5 / 5

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CRISPR/Cas9 knockout of USP18 enhances type I IFN responsiveness and restricts HIV‐1 infection in macrophages.It is well established that type I IFNs (T1 IFNs) can restrict acute HIV‐1 infection in vitro.1, 2, 3, 4, 5 In clinical trials treating human patients with recombinant IFN‐α2a, T1 IFNs can suppress viral replication in the absence of antiretroviral therapy (ART) in some patients.6, 7 However, this approach failed in long‐term treatment when study subjects became refractory to IFN treatment and viral loads returned to previous levels.

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ISG15 has also been shown to regulate type I interferon signalling by stabilizing USP18 (ref.

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The impact of HIRI on LCMV replication (see Fig. 6A and B) was evaluated by one-way analysis of variance with the Tukey's post hoc test.We have previously shown that USP18 can modulate the type 1 IFN response (14).

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USP18 affects JAK

| 7

USP18 inhibits JAK.

| 5

USP18 inhibits JAK. 5 / 9

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Taken together, we concluded that USP18 potentiates the anti-HBV activity of IFN-alpha by targeting the JAK and STAT signaling pathway in Hepg2.2.15 cells.

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As expected, exogenous USP18 expression significantly inhibited IFN-a-induced Jak and STAT signaling as shown by decreased p-STAT1 levels and ISRE activity.

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Moreover, USP18 competitively inhibits IFN-alpha and beta-induced JAK and STAT activation [XREF_BIBR] and upregulates epidermal growth factor receptor (EGFR) expression [XREF_BIBR].

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USP18 activates JAK.

| 2

USP18 activates JAK. 2 / 3

| 2

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In contrast, silencing USP18 activates the Jak and STAT signaling and potentiates IFN anti-HCV ativity [XREF_BIBR].

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USP18 affects KRAS

| 12

USP18 activates KRAS.

| 6

USP18 activates KRAS. 5 / 5

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Repression of USP18 not only decreased KRAS protein stability, but also conferred KRAS mislocalization from the plasma membrane to the endomembrane compartment.

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Since in vitro data revealed USP18 knockdown decreased KRAS protein stability it was sought to learn if Usp18 loss (XREF_SUPPLEMENTARY) affected lung cancer formation in the Kras driven mouse model.

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Using the protein synthesis inhibitor cycloheximide (CHX), USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability.

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Using the protein synthesis inhibitor cycloheximide, USP18 knockdown significantly reduced the half-life of KRAS, but gain of USP18 expression significantly increased its stability.

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Loss of USP18 Reduces Tumorigenicity of Kras driven Lung Cancers in Mice.

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USP18 activates mutated KRAS. 1 / 1

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These findings showed that reduced USP18 levels can antagonize growth of KRAS mutant lung cancers, Yet, destabilization of ISGylated proteins downstream of KRAS might also be responsible for the growth inhibition observed after USP18 loss.

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USP18 decreases the amount of KRAS.

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Modified USP18 decreases the amount of KRAS. 2 / 2

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Restoration of USP18 levels in USP18 repressed cells augmented KRAS expression versus vector controls (XREF_FIG).

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Interestingly, loss of USP18 reduced KRAS expression, and engineered gain of USP18 expression increased KRAS protein levels in lung cancer cells.

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USP18 decreases the amount of KRAS. 1 / 1

| 1

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USP18 inhibits mutated KRAS. 1 / 1

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Gain of USP18 expression enhanced (XREF_FIG) and loss of USP18 significantly (P < 0.05) reduced (XREF_FIG and XREF_SUPPLEMENTARY) growth of KRAS mutant lung cancer cell lines.

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USP18 affects cell growth

| 1 9

USP18 activates cell growth.

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USP18 activates cell growth. 7 / 8

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Likewise, engineered loss of USP18 expression decreased lung cancer cell growth and increased apoptosis in these cancer cells [XREF_BIBR and LM Mustachio personal communication].

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USP18 promotes PC cell growth in vitro and in vivo.

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To further understand the mechanism by which USP18 contributes to PC cell growth, we investigated the effects of USP18 knockdown on the cell cycle and apoptosis.

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Subsequently, we demonstrated that knockdown of USP18 inhibited HCC cell growth and caused cell-cycle arrest and early apoptosis.

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USP18 promotes PC cell growth by facilitating cell cycle progression.

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Consonant with our observation of enhanced cell proliferation of HEK293 and M15 cells upon exogenous Usp18 expression, a similar promotion of cellular growth by Usp18 has recently been reported in an acute promyelocytic leukemic cell lines [XREF_BIBR].

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Another study showed that USP18 expression was increased in malignant versus normal lung tissue, and loss of USP18 expression decreased lung cancer cell growth and increased apoptosis in these cancer [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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USP18 inhibits cell growth.

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USP18 inhibits cell growth. 3 / 3

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For instance, downregulation of USP18 reduces acute promyelocytic leukaemia cell growth and induces apoptosis, whereas silencing USP18 in glioblastoma cells enhances IFN induced apoptosis [XREF_BIBR].

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The CCK8 and EdU assay data showed that silencing USP18 obviously suppressed pancreatic cancer cell growth in vitro (XREF_FIG, XREF_FIG).

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Subsequently, we demonstrated that knockdown of USP18 inhibited HCC cell growth and caused cell-cycle arrest and early apoptosis.

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USP18 affects STAT1

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USP18 inhibits STAT1. 8 / 10

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USP18 negatively regulates the activation of STAT1 upon interaction with IFNAR2 .

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Silencing of USP18 by specific siRNA attenuated the pSTAT1 suppression by Ach.

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XREF_BIBR, XREF_BIBR To asses whether STAT1 or STAT2, which are both upregulated by USP18 inhibition (XREF_FIG), are implicated in the upregulation of Bim, DP5 and PUMA mRNA expression in USP18 silenced cells, we inhibited in parallel USP18 and STAT1 or STAT2 in INS-1E cells by use of specific siRNAs.

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We further found that microglial Usp18 negatively regulates the activation of Stat1 and concomitant induction of interferon induced genes, thereby terminating IFN signaling.

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Therefore, knockdown USP18 leads to prolonged and enhanced the activity of STAT1, and upregulated expression of many ISGs.

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Silencing USP18 prolongs the phosphorylated state of signal transducer and activator of transcription 1 (STAT1) and enhances the expression of ISGs in response to IFN-alpha [XREF_BIBR].

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USP18 negatively regulates the activation of STAT1 upon interaction with IFNAR2 (Malakhova et al., 2006).

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Of note, USP18 knockdown did not increase pSTAT1 in response to IFN-alpha before 24 h; these findings are consistent with previous observations.

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USP18 bound to IFNA inhibits STAT1. 1 / 1

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The binding of USP18 with the IFN-α receptor has been shown to inhibit the interaction of STAT-1 with the IFN-α receptor and thus block downstream IFN signaling (12, 15).

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IL10 affects USP18

| 1 9

IL10 increases the amount of USP18.

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IL10 increases the amount of USP18. 8 / 9

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In the present study, treatment of hepatic cells with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression in hepatocytes.

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USP18 mRNA expression was induced by TNF-α and LPS but not by IL-6 or IL-10 (Fig. 1A).

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Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-α refractory state, which was reversed by USP18 knockdown.

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We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-α response.

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USP18 mRNA expression was induced by TNF-alpha and LPS but not by IL-6 or IL-10 (XREF_FIG).

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In the present study, treatment of hepatic cells with LPS and TNF-alpha, but not IL-6 or IL-10, led to upregulated USP18 expression in hepatocytes.

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Treatment of Huh7.5 cells and primary murine hepatocytes with LPS and TNF-alpha, but not IL-6 or IL-10, led to upregulated USP18 expression and induced an IFN-alpha refractory state, which was reversed by USP18 knockdown.

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We examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-6 (IL-6) and IL-10 to upregulate hepatocyte USP18 expression and blunt the IFN-alpha response.

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IL10 activates USP18.

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IL10 activates USP18. 2 / 2

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USP18 is induced in hepatocytes by LPS and TNF - but not by IL-6 and IL-10 .

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USP18 is induced in hepatocytes by LPS and TNF-alpha but not by IL-6 and IL-10.

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USP18 affects IL2

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USP18 inhibits IL2.

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USP18 inhibits IL2. 4 / 6

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Genetic depletion of USP18 causes NF-κB and NFAT hyperactivation and hyperproduction of IL-2 in T cells [154].

| PMC

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To understand the molecular mechanisms by which USP18 reduces IL-2 production in T cells, we first examined the activation of TCR proximal signaling events.

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Taken together, USP18 downregulates IL-2 synthesis and TCR induced T cell proliferation [XREF_BIBR].

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Taken together , USP18 downregulates IL-2 synthesis and TCR-induced T cell proliferation [ 43 ] .

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USP18 increases the amount of IL2.

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USP18 increases the amount of IL2. 2 / 2

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USP18 activates MAP3K7. 6 / 6

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However, NEMO deficiency did not lead to loss of interaction between USP18 and TAK1 (XREF_FIG), suggesting that USP18 targets TAK1 complex and IKK complex by distinct mechanisms.

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Based on our experimental data, we propose a working model to explain how USP18 negatively regulates NF-kappaB signaling by targeting TAK1 and NEMO.

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After TLR ligand stimulation, TAK1 and NEMO undergo K63 linked ubiquitination by TRAF6, while upregulated USP18 targets TAK1 and NEMO.

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USP18 negatively regulates NF-kappaB signaling by targeting TAK1 and NEMO for deubiquitination through distinct mechanisms.

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Considering the phenotypes observed with Tak -/- and Usp18 -/- T cells or Usp18 -/- T cells transfected with USP18 (WT) or USP18 (C61S), we further hypothesized that USP18 targets TAK1 complex and catalyzes deubiquitination of TAK1.

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Recent work has demonstrated that USP18 has the ability to deubiquitinate the transforming growth factor-activated kinase 1 (TAK1) complexes required for NF-κB activation in T cells and that overexpression of USP18 leads to decreased nuclear activation and impaired formation of TAK1 complexes (47).

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USP18 inhibits MAP3K7.

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USP18 inhibits MAP3K7. 1 / 4

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Recent work has demonstrated that USP18 has the ability to deubiquitinate the transforming growth factor activated kinase 1 (TAK1) complexes required for NF-kappaB activation in T cells and that overexpression of USP18 leads to decreased nuclear activation and impaired formation of TAK1 complexes.

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IL6 affects USP18

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IL6 increases the amount of USP18.

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IL6 increases the amount of USP18. 6 / 7

| 6

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In the present study, treatment of hepatic cells with LPS and TNF-alpha, but not IL-6 or IL-10, led to upregulated USP18 expression in hepatocytes.

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USP18 mRNA expression was induced by TNF-α and LPS but not by IL-6 or IL-10 (Fig. 1A).

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USP18 mRNA expression was induced by TNF-alpha and LPS but not by IL-6 or IL-10 (XREF_FIG).

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In the present study, treatment of hepatic cells with LPS and TNF-α, but not IL-6 or IL-10, led to upregulated USP18 expression in hepatocytes.

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IL6 activates USP18.

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IL6 activates USP18. 3 / 3

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USP18 is induced in hepatocytes by LPS and TNF-alpha but not by IL-6 and IL-10.

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USP18 is induced in hepatocytes by LPS and TNF - but not by IL-6 and IL-10 .

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The cytokine specificity of our results is also consistent with finding that IL-6 alone is not able to induce USP18 in murine T cells.

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IFNB1 affects USP18

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IFNB1 increases the amount of USP18.

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IFNB1 increases the amount of USP18. 4 / 5

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77,78 IFN-β can induce USP18 expression through IFNAR.

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On the other hand, the upregulation of USP18 inhibits beta cell apoptosis.75, 76IFN-β is considered the first line of therapy against MS.77, 78 IFN-β can induce USP18 expression through IFNAR.

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Finally, AA homozygosis for the intronic polymorphism rs2542109 was associated with the responder phenotype; however, USP18 expression levels induced by IFNbeta did not differ amongst MS patients carrying different rs2542109 genotypes.

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XREF_BIBR, XREF_BIBR MS. IFN-beta is considered the first line of therapy against MS. XREF_BIBR, XREF_BIBR IFN-beta can induce USP18 expression through IFNAR.

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IFNB1 activates USP18.

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IFNB1 activates USP18. 4 / 5

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16 USP18 is induced by IFN-beta not only in lymphocytes but also in HO-1 human melanoma cells, 6 in Huh-7.5 cells treated with irrelevant small interfering RNAs, 17 in the choroid plexus and in ependymal cells.

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A previous study showed that USP18 is induced by IFN-beta in HO-1 human melanoma cells XREF_BIBR.

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In contrast, Li et al. (2000) demonstrate that ISG43 (HuUBP43) is induced maximally by IFN-beta, in 2fTGH cells, whereas IFN-gamma (200 U/ml) is a better inducer of this gene than IFN-alpha (500 U/ml)[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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16 USP18 is induced by IFN-β not only in lymphocytes but also in HO-1 human melanoma cells, 6 in Huh-7.5 cells treated with irrelevant small-interfering RNAs, 17 in the choroid plexus and in ependymal cells.

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WT1 affects USP18

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WT1 decreases the amount of USP18.

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WT1 decreases the amount of USP18. 6 / 6

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A variety of subsequent, in vitro experiments consistently supported the notion that WT1 suppresses Usp18 expression, and we therefore conclude that Usp18 is a bona fide target for WT1 transcriptional regulatory function in the embryonic kidney.

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Despite this increased expression, this truncated WT1 still was unable to repress the expression of the Usp18 reporter construct.

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Taken together our data demonstrate that Usp18 is a transcriptional target of WT1 and suggest that increased expression of USP18 following WT1 loss contributes to Wilms tumorigenesis.

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These two independent experiments suggest that WT1 represses Usp18 expression in mammalian cells.

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Taken together our data suggest that increased expression of USP18 following WT1 loss contributes to Wilms tumorigenesis.

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Cumulatively, these data indicate that WT1 (-KTS) down-regulates Usp18 expression via direct or indirect interaction with the Usp18 promoter.

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SKP2 inhibits USP18. 5 / 5

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We therefore analysed the impact of ISG15 on SKP2 mediated USP18 degradation.

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This binding prevents the SKP2 mediated degradation of USP18, and thus is critical for the accumulation of USP18 and the appropriate regulation of IFN-alpha and beta signalling.

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ISG15 was shown to bind to and prevent uSP18 degradation mediated by S-phase kinase-associated protein 2 (SKP2)-dependent ubiquitylation 4,114 .

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In humans, binding of free ISG15 prevents proteasomal degradation of USP18 by SKP2 (Tokarz et al., 2004) and is critical to ensure negative regulation of IFN-α/β immunity by stabilizing USP18 (Zhang et al., 2015).

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In humans , binding of free ISG15 prevents proteasomal degradation of USP18 by SKP2 ( Tokarz et al ., 2004 ) and is critical to ensure negative regulation of IFN-alpha / beta immunity by stabilizing USP18 ( Zhang et al ., 2015 ) .

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SKP2 activates USP18.

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As summarized above, SKP2 can target USP18 for degradation, while ISG15 has an opposite action, being required for accumulation of USP18 as seen in IFN stimulated cells.

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Patient fibroblasts, transduced with control luciferase (Luc) or with USP18, were treated with IFNalpha and SKP2 was immunoprecipitated.

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SKP2 (S-phase kinase associated protein 2) was found to target USP18 to ubiquitination and proteosomal degradation 10.

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IFNG affects USP18

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IFNG activates USP18.

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2 In addition, USP18 has been found to inhibit TRAIL-induced apoptosis independently of the deISGylation pathway.91 Furthermore, the depletion of USP18 leads to a strong increase in the levels and activity of miR-7, and this activity in turn decreases the expression of EGFR, leading to apoptosis and control of cancer cells.92Hong et al.93 found that IFN-γ can induce USP18 in tumor cells and that this protein plays an important role in inhibiting tumorigenesis and maintaining antitumor immunity.

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The novel finding in this study is the discovery that the ubiquitin specific peptidase USP18 can be induced by IFN-gamma in tumor cells and plays important roles in inhibiting tumorigenesis and antitumor immunity.

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92 Hong et al. 93 found that IFN-γ can induce USP18 in tumor cells and that this protein plays an important role in inhibiting tumorigenesis and maintaining antitumor immunity.

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The low-level induction of USP18 by IFN-gamma was not sufficient to attenuate (peg) IFN-alpha-mediated signaling.

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92 Hong et al. 93 found that IFN-gamma can induce USP18 in tumor cells and that this protein plays an important role in inhibiting tumorigenesis and maintaining antitumor immunity.

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IFNG increases the amount of USP18.

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IFN-gamma signaling induces USP18 expression in tumor cells during immunosurveillance.

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In this report, we investigated the function of USP18 in IFN-gamma signaling in B16 melanoma cells in vitro and in vivo and found that IFN-gamma or CTLs activated USP18 expression in tumor cells.

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USP18 expression in tumor cells, such as human sarcoma 2fTGH cells, is not only induced by IFNgamma timulation [XREF_BIBR].

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In conclusion, we found that IFN-gamma signaling induces intrinsic expression of USP18 in tumor cells that not only affects tumorigenesis, but also may be useful in regulating immunotherapy efficacy.

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USP18 affects USP18

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USP18 activates USP18.

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In GM-CSF-supplemented culture, over-expression of Usp18 restored the development of CD11b + CD11c + cells in Usp18 deficient BM cells, but did not affect DC development in wild-type BM cells (XREF_FIG), which confirmed the notion that the development defect of BM-DCs is intrinsic to Usp18 deficient cells.

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Usp18 (-/-) BM cells were rescued by exogenous expression of either wild-type or deconjugation-inactive Usp18, and superimposition of an IFN-alpha and beta 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.

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USP18 bound to IRS4 activates USP18. 1 / 1

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IRS4 binding to USP18 diminished the inhibitory effect of USP18 on Jak and STAT signaling.

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USP18 bound to IFNAR2 activates USP18. 1 / 1

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USP18 decreases the amount of USP18.

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USP18 was revealed to be upregulated in lung cancer, and reduced USP18 expression was found to be associated with significantly longer cancer specific survival in patients with muscle-invasive bladder cancer [XREF_BIBR, XREF_BIBR].

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USP18 affects cell cycle

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USP18 activates cell cycle.

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USP18 activates cell cycle. 6 / 6

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Consistently, the loss of c-Myc reversed the cell cycle arrest and apoptosis induced by USP18 overexpression in SW1990 cells (XREF_FIG - XREF_FIG).

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Moreover , flow cytometry showed that downregulation of USP18 significantly arrested the cell cycle in G1 phase in pancreatic cancer cells .

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Moreover, our experimental data revealed that USP18 silencing obviously blocked cell cycle at G1 phase and increased cell apoptosis.

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Cell cycle analysis showed that USP18 silencing caused a considerable inhibition of cell cycle progression, leading to a selective accumulation of cells in the G1 phase compared with control groups in[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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Overall, these results suggested that USP18 promotes pancreatic cancer cell proliferation by facilitating cell cycle progression and inhibiting cell apoptosis.

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USP18 promotes PC cell growth by facilitating cell cycle progression.

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USP18 inhibits cell cycle.

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USP18 inhibits cell cycle. 2 / 2

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Moreover, flow cytometry showed that downregulation of USP18 significantly arrested the cell cycle in G1 phase in pancreatic cancer cells.

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In present study, our data indicated that knockdown of USP18 could inhibit cell proliferation and induce cell cycle arrest in the G1 phase in HCC cells.

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SGCG affects USP18

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SGCG activates USP18.

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Firstly, like ISG15 itself, the predominant ISG15 conjugation and deconjugation enzymes UbE1L, UbcH8/UbcM8, HERC5/HERC6, and UBP43/USP18 are all induced by type I IFN stimulation.

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The cloning of USP18 was also independently reported by 3 other groups in various species, and all confirmed the induction of USP18 by Type 1 IFN (Zhang and others 1999; Li and others 2000; Kang and others 2001) , consistent with the finding of highly conserved ISREs in the UBP43 promoter (Malakhova and others 2002) .

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Further temporal regulation of the ISG15 system by expression of the type I IFN-induced cellular deconjugase (human Usp18/mouse Ubp43), as well the role of free intracellular ISG15 in downregulating signaling at the type I IFN receptor in human cells, remains to be explored.We have shown that ISG15 is secreted from both lymphocytes and epithelial cells, suggesting that the ISG15 secretion mechanism is operational in a wide range of cell types.

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b | Intracellular functions: Ubl carboxy-terminal hydrolase 18 (USP18) and S-phase kinase-associated protein 2 (SKP2): USP18, which is induced by type I interferons, mediates the negative feedback regulation of interferon signalling independent of its deISGylase activity.

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SGCG increases the amount of USP18.

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The expression of USP18 is strongly induced by type I and type III interferons [2] [3] [4] [5] , by the Toll-like receptor (TLR) agonists LPS [6] [7] [8] and polyI:C [6] (synthetic analogy of dsRNA) and by TNFα [7] .

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The expression of USP18 is strongly induced by type I and type III interferons [2–5], by the Toll-like receptor (TLR) agonists LPS [6–8] and polyI:C [6] (synthetic analogy of dsRNA) and by TNFα [7].

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IFNL4 affects USP18

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IFNL4 increases the amount of USP18.

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STAT1 activates USP18.

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STAT1 activates USP18. 3 / 3

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The second mechanism is the recently described inhibition of STAT1 phosphorylation by UBP43 (the product of ISG43) through its inhibition of JAK1 interaction with the IFNAR2 subunit of the type I IFN receptor XREF_BIBR.

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STAT1 , USP18 and IRF1 modulate CXCL10 levels in EC following IFN-alpha stimulation To validate the transcriptional regulations inferred in our multiple-output FFL regulatory subnetwork ( Fig. 2 ) , we measured CXCL10 protein levels in tissue culture media conditioned by STAT1 - , USP18 - , IFIH1 - and IRF1-silenced HUVECs , compared to HUVECs transfected with a scrambled siRNA ( siCTRL ) .

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This study showed that type I IFNs (but not type II IFNs) were required for CD95 induced stemness and did so through the phosphorylation and activation of STAT1 and upregulation of the STAT1 targets PLSCR1, USP18, and HERC8.

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This immunological response is explained by the impairment of STAT4 phosphorylation and IFN-γ production as the result of STAT1 hyperactivation.11 Another group of researchers attributed the susceptibility of Usp18lty9 mice to S. typhimurium or Mycobacterium tuberculosis infection to elevated levels of IL-10, IL-1β, or IL-17, in addition to the deregulation of autophagy markers.10 Surprisingly, the contradictory nature of these two results obtained with Usp18 knockout mice and Usp18lty9 mice can be explained by either the different backgrounds of the mice or the specific ENU-induced mutation of Usp18.

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STAT1 inhibits USP18.

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Of note, we did not find evidence that STAT1 mediates the USP18 dependent reduction in neurogenesis.

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USP18 activates signal transduction.

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211, 214 ISG15, together with its conjugation E3 ligase (CEB1) and its deconjugation enzyme USP18, are in the same ISG15/USP18 UBL pathway.ISGylation modulates signal transduction pathways and host antiviral responses.

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Suppressive pathways include IFN-I activation of USP18, an ISG that suppresses signal transduction by reducing the ability of IFN-Is to form an active receptor complex (38, 48).

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USP18 inhibits signal transduction.

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The non-covalent interactions of ISG15 and USP18 prevent the ubiquitination of USP18 by S-phase kinaseassociated protein two and stabilize the downregulation of the IFN signaling pathway by USP18 (Tokarz et al., 2004; Zhang et al., 2015) .

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Interestingly, USP18 has been shown to negatively regulate the type I IFN signalling pathway, and its deficiency results in enhanced and prolonged STAT1 phosphorylation XREF_BIBR - XREF_BIBR.

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USP18 can interact with IFNAR2 and STAT2, competing with JAK1 for receptor binding and thus inhibiting signal transduction.

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USP18 affects CD8

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USP18 inhibits CD8.

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Furthermore, in comparison to control mice, the frequency of conventional CD11b + DCs in the spleen of Usp18 -/- mice was reduced by about 50% (XREF_FIG), however, the conventional CD8 + DCs (XREF_FIG) and pDCs (CD11c int B220 + CD11b -) (data not shown) populations were observed with the same frequency in the spleens of both Usp18 -/- and control mice.

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In conclusion, lack of Usp18 in CD11c + cells reduced priming of islet specific CD8 + T cells and prevented induction of diabetes.

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We found here that lack of Usp18 in dendritic cells prevented enforced virus replication and would therefore limit induction of autoreactive CD8 + T cells, but also induction of IFN-I production.

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The absence of Usp18 limited the expansion of virus specific CD8 + T cells (XREF_FIG) and reduced IFN-gamma production by CD8 + T cells (XREF_FIG) and CD4 + T cells (XREF_FIG).

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USP18 activates CD8.

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(b) In case of enforced viral replication in dendritic cells of a virus resembling an autoantigen, autoreactive CD8 + T cells will be primed, which leads to autoimmune diseases such as type I diabetes, whereas USP18 deficiency reduces the priming of autoreactive CD8 + T cells and onset of autoimmune diabetes owing to inhibition of enforced viral replication Functions of USP18 N Honke et al recognized as essential player for the development of MS.

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This finding suggests that, in the presence of virus specific antibodies, Usp18 is necessary for viral replication in marginal zone macrophages and also enhances the priming of virus specific CD8 + T cells.

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Therefore we would suggest that Usp18 expression in dendritic cells could drive autoimmune diabetes by promoting activation of cross-reactive CD8 + T cells, but also by induction of high levels of IFN-I.

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USP18 affects PTEN

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USP18 inhibits PTEN.

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USP18 inhibits PTEN. 2 / 2

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It was therefore not surprising that repression of USP18 augmented PTEN cytoplasmic destabilization.

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Data displayed here indicate that loss of USP18 induced destabilization of PTEN protein in the cytoplasm.

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Modified USP18 inhibits PTEN. 1 / 1

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This finding established that loss of USP18 expression reduced cellular PTEN levels and promoted destabilization of cytosolic PTEN [XREF_BIBR, XREF_BIBR].

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USP18 activates PTEN.

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USP18 activates PTEN. 2 / 2

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However, USP18 overexpression could stabilize PTEN protein, and USP18 repression decreases mainly cytoplasmic PTEN [XREF_BIBR].

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Intriguingly, enhanced ISGylation in these mice mediated increased resistance against influenza and vaccinia virus infections and diminished myocarditis upon cocksackie virus B3 infection, thus qualifying USP18 protease inhibition as a potential antiviral strategy xref , xref .

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Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication.

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Thus, boosting ISG15 modification by protease inhibition of USP18 might represent a new strategy to interfere with viral replication.

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This mouse model shows enhanced ISGylation levels because of the USP18 protease inactivation whereas they do not show apparent phenotypic alterations (Ketscher et al., 2015).

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This mouse model shows enhanced ISGylation levels because of the USP18 protease inactivation whereas they do not show apparent phenotypic alterations .

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USP18 affects inflammatory response

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USP18 reduced basal inflammation, senescence and insulin resistance in coronary endothelial cells.

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From the transcriptomic analysis we selected USP18, previously shown to decrease inflammation and insulin-resistance.

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Our findings indicate that USP18 inhibition induces inflammation by increasing the STAT signaling and exacerbates IFN induced beta cell apoptosis by the mitochondrial pathway of cell death.

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USP18 silencing enhanced basal inflammation and senescence.

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USP18 activates inflammatory response.

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USP18 interacts with IFNAR2 to prevent JAK1 from interacting with IFNAR2, and thus, USP18 suppresses signal transmission from IFN-alpha binding [XREF_BIBR, XREF_BIBR].

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2,3,7,8-tetrachlorodibenzodioxine affects USP18

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2,3,7,8-tetrachlorodibenzodioxine increases the amount of USP18.

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2,3,7,8-tetrachlorodibenzodioxine decreases the amount of USP18.

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USP18 activates TNFSF10.

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USP18 activates TNFSF10. 4 / 4

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23 A study using an oncogenic cell line (E1A cells) found that USP18 activates the extrinsic TNF-related apoptosis-inducing ligand (TRAIL) pathway after IFN-α challenge.

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23 A study using an oncogenic cell line (E1A cells) found that USP18 activates the extrinsic TNF related apoptosis inducing ligand (TRAIL) pathway after IFN-alpha challenge.

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Treating Usp18 -deficient hematopoietic cells with Poly I:C decreases the number of white blood cells since apoptosis is not prevented by USP18. xref Moreover, knocking down USP18 markedly enhances the NF- κ B signaling induced by various TLR ligands. xref A study using an oncogenic cell line (E1A cells) found that USP18 activates the extrinsic TNF-related apoptosis-inducing ligand (TRAIL) pathway after IFN- α challenge. xref In human promonocytic THP-1 cells, the expression of proinflammatory cytokines such as TNF- α , interleukin-6 (IL-6), and IL-1 β is significantly higher when USP18 is silenced with siRNA. xref Interestingly, in contrast to E1A cells, Usp18 -deficient murine bone marrow cells and THP-1 cells that have been treated with IFN α / β do not experience apoptosis after treatment with TRAIL or FASL. xref However, IFN- α / β still triggers apoptosis in these cells through the mitochondrial pathway and the reactive oxygen species pathway, xref a finding indicating that USP18 influences cell survival in various pathways depending on the cell type.

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USP18 decreases the amount of TNFSF10.

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For instance, deletion or reduction of USP18 gene suppresses cell proliferation and induces cell apoptosis by inducing the expression of TRAIL [11].

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USP18 affects Neoplasm Metastasis

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USP18 activates Neoplasm Metastasis.

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USP18 activates Neoplasm Metastasis. 4 / 4

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USP18 promotes tumor metastasis in esophageal squamous cell carcinomas via deubiquitinating ZEB1.

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USP18 promotes tumor metastasis in esophageal squamous cell carcinomas via deubiquitinating ZEB1 .

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In functional experiments , USP18 knockdown significantly inhibited ESCC invasion and metastasis in vitro .

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In functional experiments, USP18 knockdown significantly inhibited ESCC invasion and metastasis in vitro.

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USP18 inhibits Neoplasm Metastasis.

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USP18 inhibits Neoplasm Metastasis. 1 / 1

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Interestingly, lack of Usp18 reduced the incidence of lung metastasis in PyVmT mice (XREF_FIG) that could be related to a decrease in invasiveness of cancer cells observed in in vitro matrigel invasion assays (XREF_FIG).

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USP18 affects Neoplasm Invasiveness

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USP18 activates Neoplasm Invasiveness.

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In functional experiments , USP18 knockdown significantly inhibited ESCC invasion and metastasis in vitro .

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At necropsy 6 weeks post-infection, Usp18 Ity9 mutant mice showed large infected foci with extensive necrotic centre and increased lymphohistiocytic inflammatory cell infiltration in the lung compared to littermate control mice with a greater percentage of the lung affected by inflammation (XREF_SUPPLEMENTARY, left and middle panels).

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In functional experiments, USP18 knockdown significantly inhibited ESCC invasion and metastasis in vitro.

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USP18 inhibits Neoplasm Invasiveness.

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Functionally, decreased USP18 expression attenuated GBM cell invasion and migration through repressing EMT.

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Lack of Usp18 inhibits angiogenesis and reduces invasiveness of mammary epithelial tumour cells.

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USP18 affects IFNAR1

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USP18 inhibits IFNAR1.

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For example, USP18 is known to prevent IFNAR activation as shown by Honke et al. where USP-18 secreting CD169+ macrophages displayed a lower type I IFN sensitivity upon viral infection [38], [57].

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For example, USP18 is known to prevent IFNAR activation as shown by Honke et al. where USP-18 secreting CD169 + macrophages displayed a lower type I IFN sensitivity upon viral infection [38, 57] .4.

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Lacking of USP18 leads to an increase signaling of IFN-I, IFN-III, TNF-α and high levels of conjugated ISG15Figure 3: Role of USP18-dependent enforced viral replication in activation of the adaptive immune system and onset of diabetes mellitus type I. (a) USP18 inhibits IFNAR signaling, which leads to enforced viral replication in CD169+ macrophages.

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Th17 cells are widely Figure 3 Role of USP18-dependent enforced viral replication in activation of the adaptive immune system and onset of diabetes mellitus type I. (a) USP18 inhibits IFNAR signaling, which leads to enforced viral replication in CD169 + macrophages.

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USP18 activates IFNAR1.

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Thus, although CD169+ macrophages in both spleen and LN preferentially allow VSV replication, whether Usp18 mediates downmodulation of IFNAR signaling in LN SCS macrophages needs to be examined to complete the circuit.

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USP18 affects Protease

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USP18 activates Protease.

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A novel interferon stimulated gene encoding a 43-kDa ubiquitin specific protease, designated ISG43, was identified in this screen.

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The IFN stimulated gene ubiquitin specific proteinase 18 (USP18) encodes a protein that negatively regulates T1 IFN signaling via stearic inhibition of JAK1 recruitment to the IFN-alpha receptor 2 subunit (IFNAR2).

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USP18 inhibits NOTCH1.

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USP18 inhibits NOTCH1. 3 / 3

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Moreover, USP18 overexpression reduced the K48 linked ubiquitination level of Notch1, whereas USP18 silencing significantly increased Notch1 K48 linked ubiquitination (XREF_FIG - XREF_FIG).

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Indeed, we found here that USP18 silencing could increase the turnover rate of Notch1 in the presence of cycloheximide (CHX) (XREF_FIG and XREF_FIG).

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USP18 activates MYC. 3 / 3

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To further validate that USP18 mediated the growth of PC cells by regulating c-Myc, we first increased the expression of c-Myc in USP18 knockdown PC cells and then measured the USP18 and c-Myc protein expression levels and cell proliferation.

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USP18 null leiomyosarcoma cell lines are aneuploid and overexpress MYC.

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USP18 decreases the amount of CXCL10.

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USP18 decreases the amount of CXCL10. 3 / 3

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These results were confirmed in primary rat beta cells (XREF_FIG) in which USP18 inhibition upregulated CXCL10, CCL5 and IL-15 mRNA expression.

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We demonstrated that USP18 deficient cells have increased expression of IFIT1, IFIT2, IFIT3, IFITM1, IFITM2, CXCL9, CXCL10, ISG15, and MX2 after treatment with exogenous IFN‐β.

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USP18 increases the amount of CXCL10.

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USP18 increases the amount of CXCL10. 1 / 1

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Hypersensitivity of PyVmT and Usp18 KO MECs to IFN-lambda enhances upregulation of Cxcl10 expression and inhibits tumour progression.

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USP18 activates CXCL10.

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We next determined if iMacs can support HIV-1 replication as previously reported 48 and if USP18 is induced by HIV-1 in iMacs .

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USP18 affects SKP2

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USP18 activates SKP2.

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USP18 activates SKP2. 3 / 3

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For instance, Tan et al. found that USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway [XREF_BIBR].

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USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway.

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Tan et al. have demonstrated that USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway.

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USP18 increases the amount of SKP2.

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Modified USP18 increases the amount of SKP2. 1 / 1

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Our main observation is that forced expression of USP18 and ISG15 modulate the level of SKP2, whether endogenous or overexpressed.

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USP18 affects Infections

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USP18 inhibits Infections.

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USP18 inhibits Infections. 3 / 3

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Therefore, these results suggested that inhibiting the expression of IFIT3, OASL, USP18, XAF1, IFI27, and EPSTI1 may reduce the risk of SARS‐CoV‐2 infection and may also have a positive effect on antiviral therapy in patients with SARS‐CoV‐2 infection.In conclusion, this study comprehensively analyzed the blood leukocytes gene expression profile data of COVID‐19 patients by using bioinformatics methods and provided a preliminary understanding of the functions and mechanisms of DEGs in the leukocytes of COVID‐19 patients.

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Therefore, these results suggested that inhibiting the expression of IFIT3, OASL, USP18, XAF1, IFI27, and EPSTI1 may reduce the risk of SARS-CoV-2 infection and may also have a positive effect on antiviral therapy in patients with SARS-CoV-2 infection.and 4G).

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This will also induce the expression of USP18, an inhibitor of the IFN signaling pathway, leading to an impaired antiviral state and to an increased propensity to develop chronic infection.

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USP18 activates Infections.

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USP18 activates Infections. 1 / 1

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We speculate that by blocking IFN-α signaling, USP18 expression may lead to an enhanced susceptibility to infection with interferon-sensitive viruses and enhanced viral proliferation.

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USP18 affects IFNB1

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USP18 inhibits IFNB1.

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6 In turn, USP18 inhibits IFN-β signaling.

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USP18 activates AKT. 3 / 3

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USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway.

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Tan et al. have demonstrated that USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway.

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For instance, Tan et al. found that USP18 promotes breast cancer growth by upregulating EGFR and activating the AKT and Skp2 pathway [XREF_BIBR].

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USP18 inhibits AKT.

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Finally, our data demonstrate that higher USP18 in Mem from infected patients prevent proper cell survival and long lasting maintenance in an AKT dependent manner.

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TLR affects USP18

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TLR increases the amount of USP18.

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Furthermore, TLR ligands, LPS, Pam3CSK4 and CL097 all gave the same result of increased mRNA level of USP18, supporting that TLR-induced signaling pathway induces USP18 expression [42].

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Other TLR ligands, such as Pam3CSK4 (TLR2 ligand) or CL097 (TLR7/8 ligand), could also induce USP18 expression in THP-1 cells (XREF_FIG).

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These results indicate that USP18 expression can be induced by TLR induced signaling pathways, which, in turn, can suppress TLR mediated NF-kappaB activation to form a negative feedback loop.

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TLR activates USP18.

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USP18 was upregulated by various TLR ligands in THP-1 (a human monocyte cell line) cells and inhibited IkappaB degradation as well as NF-kappaB activation to form a negative feedback loop.

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Infections affects USP18

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Infections increases the amount of USP18.

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Infections activates USP18.

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In a recent study, Ye et al. (2021) showed that USP18 induced by DENV-2 infection is a critical host factor used by DENV-2 to antagonize IFN-α production.

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11 Moreover, infection with S. typhimurium enhances IFN-I signaling and inflammatory response in Usp18 lty9 mice.

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17beta-estradiol affects USP18

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17beta-estradiol increases the amount of USP18.

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17beta-estradiol decreases the amount of USP18.

However, it should be noted that in this study, USP18 expression in tumor cells not only affected tumor cell activity, but also regulated immune cells in tumor microenvironment in that tumor cell USP18 expression also activated CD11c + dendritic cells residing in the tumor.

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Only virus infection, supported by the Usp18 driven enforced virus replication process in CD11c + APCs is efficient in breaking immunologic tolerance to pancreatic islet cells in our model.

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USP18 affects IL10

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USP18 inhibits IL10. 2 / 3

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We show that IRF-7 siRNA knockdown enhanced LPS induced IL-10 production in human monocyte derived macrophages, and USP-18 overexpression attenuated LPS induced production of IL-10 in RAW264.7 cells.

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Supporting this hypothesis, we show that USP-18, a target of IRF-7 and known negative regulator of the type I interferon response, decreases the production of immune-regulatory cytokines IL-27 and IL-10.

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USP18 affects IKBKB

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They show that three USPs -- USP14, USP18, and USP22 -- fulfilled these criteria, but focused on USP14, as USP18 and USP22 could also inhibit IKKbeta activation directly in the absence of NLRC5.

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We found that the activation of NF-κB by MyD88, TRAF2, TRAF6, TAK1-TAB1, IKKα and IKKβ was markedly inhibited by USP18 ( xref ).

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Importantly, we observed that USP18 and USP22, but not USP14, may directly inhibit IKK-beta activation through an NLRC5 independent mechanism.

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MAP3K7 affects USP18

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After TLR ligand stimulation, TAK1 and NEMO undergo K63 linked ubiquitination by TRAF6, while upregulated USP18 targets TAK1 and NEMO.

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However, NEMO deficiency did not lead to loss of interaction between USP18 and TAK1 (XREF_FIG), suggesting that USP18 targets TAK1 complex and IKK complex by distinct mechanisms.

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Bisphenol A affects USP18

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Bisphenol A increases the amount of USP18.

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Usp18 promotes conventional CD11b + dendritic cell development.

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USP18 inhibits ITGAM.

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In addition, STAT2 CC/DB 3A, but not STAT2 CC/DB, partially blocked the effect of USP18 on transcription of ISGs, such as GBP1 and IFIT1 (XREF_FIG and XREF_SUPPLEMENTARY).

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Additionally, a homozygous miss-sense mutation in STAT2 results in failure to appropriately traffic USP18 to IFNAR2 and prevents USP18 from negatively regulating responses to IFN-Is, which leads to infant death from autoinflammation disease (168).

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USP18 affects Refractory

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It was shown that USP18 is responsible to induce this refractory state as USP18-deficient mice ( FVB background ) and cells do exhibit this kind of desensitisation after repeated injections of IFNalpha [ 5,46 ] .

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However , we have also found that USP18 is necessary but not sufficient on its own to induce an IFN - refractory state ( 53 ) .

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USP18 affects RELA

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Here, we found that ectopic expression of USP18 suppressed nuclear accumulation of p65 as well as NF-kappaB activation by LPS treatment.

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We demonstrated that USP18 deficient cells have increased expression of IFIT1, IFIT2, IFIT3, IFITM1, IFITM2, CXCL9, CXCL10, ISG15, and MX2 after treatment with exogenous IFN‐β.

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USP18 affects IRF3

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USP18 affects IL27

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USP18 inhibits IL27. 1 / 2

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USP18 affects IFNG

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IFN-lambda4 potently blocks IFN-alpha signalling by ISG15 and USP18 in hepatitis C virus infection .

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IFN-lambda4 potently blocks IFN-alpha signalling by ISG15 and USP18 in hepatitis C virus infection OPEN .

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USP18 affects Chemokine

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Further studies show that the expression of USP18 by beta islet cells themselves is important for inhibiting diabetes.75, 76 On the one hand, the upregulation of USP18 expression by IFN-I in beta islet cells prevents the activity of proinflammatory chemokines such as CCL5, CXCL10, and IL-15 and consequently inhibits insulitis.

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75, 76 On the one hand, the upregulation of USP18 expression by IFN-I in beta islet cells prevents the activity of proinflammatory chemokines such as CCL5, CXCL10, and IL-15 and consequently inhibits insulitis.

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USP18 affects CXCL9

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Carbon nanotube decreases the amount of USP18.

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USP18 inhibits cell. 1 / 1

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Induction of USP18 gene expression by candesartan would therefore inhibit interferon signaling in the cells in which this gene is expressed .

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USP18 activates cell.

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USP18 inhibits Cell Survival. 1 / 1

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Finally, our data demonstrate that higher USP18 in Mem from infected patients prevent proper cell survival and long lasting maintenance in an AKT dependent manner.

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USP18 activates Cell Survival.

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3-methylcholanthrene increases the amount of USP18. 1 / 1

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3-methylcholanthrene decreases the amount of USP18.

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USP18 affects Leu-Met

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USP18 inhibits Leu-Met. 1 / 1

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Likewise, engineered loss of USP18 expression decreased lung cancer cell growth and increased apoptosis in these cancer cells [XREF_BIBR and LM Mustachio personal communication].

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USP18 affects Leu-Asn

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Cuprizone affects USP18

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Casp14 affects USP18

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