USP25 Data Analysis

HGNC Gene Name
ubiquitin specific peptidase 25
HGNC Gene Symbol
USP25
Identifiers
hgnc:12624 NCBIGene:29761 uniprot:Q9UHP3
Orthologs
mgi:1353655 rgd:1309003
INDRA Statements
deubiquitinations all statements
Pathway Commons
Search for USP25
Number of Papers
78 retrieved on 2022-05-22

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 CCLE Correlation CCLE Z-score CCLE p-value (adj) CCLE Significant CMAP Score CMAP Type

Dependency GO Term Enrichment

Gene set enrichment analysis was done on the genes correlated with USP25using 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

Literature Mining

INDRA was used to automatically assemble known mechanisms related to USP25 from literature and knowledge bases. The first section shows only DUB activity and the second shows all other results.

Deubiquitinase Activity

psp cbn pc bel_lc signor biogrid lincs_drug tas hprd trrust ctd vhn pe drugbank omnipath conib crog dgi | rlimsp isi tees geneways eidos trips medscan sparser reach
USP25 deubiquitinates NANOG. 10 / 15
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Since USP21 is a DUB, we examined whether USP21 could directly interact with and deubiquitinate Nanog.

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USP21 deubiquitylates Nanog to regulate protein stability and stem cell pluripotency.

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USP21 directly interacts with and deubiquitinates Nanog.

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USP21 interacts with Nanog protein in embryonic stem cells in vivo and in vitro to deubiquitylate the K48-type linkage of the ubiquitin chain of Nanog, thereby stabilizing Nanog and in turn maintaining the stemness of embryonic stem cells in mice.

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USP21 deubiquitylates the K48-type linkage of the ubiquitin chain of Nanog, stabilizing Nanog.

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For example, Oct4 and Sox2 protein levels are regulated by the E3 ligase WWP2, and ubiquitin-specific-processing protease 21 (Usp21) deubiquitinates and stabilizes Nanog, which maintains pluripotency, while FBXW8, an E3 ligase, ubiquitinates and destabilizes Nanog, thereby promoting ESC differentiation.

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Considering that USP21 deubiquitylates Nanog, we sought to examine the functional roles of USP21 in mESCs.

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Next, we examined whether USP21 could deubiquitinate Nanog.

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Collectively, these data suggest that USP21 can deubiquitylate and stabilize Nanog.

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Indeed, we showed that USP21 reduced Nanog ubiquitylation directly in an in vitro deubiquitylation assay (XREF_FIG).
USP25 deubiquitinates TRAF6. 10 / 13
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Act1 mediated K63 linked ubiquitination of TRAF6 was inhibited by overexpression of USP25, but not USP25 (C178S) in 293T cells or in an in vitro deubiquitination system (XREF_FIG and XREF_SUPPLEMENTARY).

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USP25 also deubiquitinated TRAF5 and TRAF6 to regulate in IL-17 signaling [XREF_BIBR].

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Therefore, USP25 may indirectly lead to deubiquitination of TRAF5 or TRAF6 through its tightly associated proteins.

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However, Zhong et al. 's study using HEK293T cells supported deubiquitination of TRAF6 by USP25 [XREF_BIBR].

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USP25 also deubiquitinated TRAF5 and TRAF6 to regulate in IL-17 signaling [23].

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USP25 deubiquitinates RIG-I, TRAF2 and TRAF6.

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Similarly, Usp25 deubiquitinates both TRAF5 and TRAF6 and thereby restricts downstream gene expression.

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Mechanistically, USP25 deubiquitinated retinoic acid inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor mediated IFN signaling.

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However, some studies have given different results regarding USP25’s ability to deubiquitinate TRAF6.

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Mechanistically, stimulation with IL-17 induced the association of USP25 with the adaptors TRAF5 and TRAF6, and USP25 induced removal of Lys63-linked ubiquitination in TRAF5 and TRAF6 mediated by the adaptor Act2.
USP25 deubiquitinates DDX58. 10 / 11
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USP21 was also reported to antagonize the ubiquitination of anti-viral sensor RIG-I, implicating USP21 as a negative regulator of type-I interferon production and anti-viral immunity (Fan et al., 2014[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

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USP21 deubiquitinated RIG-I in HEK293 T cells [24].

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In addition, wild-type USP25 significantly inhibits ubiquitination of RIG-I, TRAF2, and TRAF6, which is essential for activation of type I IFN signaling.

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Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling.

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USP25 deubiquitinates RIG-I, TRAF2 and TRAF6.

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USP21 binds to and deubiquitinates RIG-I.

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USP17 and USP25 also deubiquitinate RIG-I in a different manner; that is, USP17 stabilizes RIG-I by K48-deubiquitination, while USP25 inhibits RIG-I degradation by K63-deubiquitination [37, 38] .

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RNA virus induced RIG-1 can be deubiquitinated by USP21 14.

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Therefore, it is highly likely that USP21 acts as a RIG-I polyubiquitination guard to prevent extensive RIG-I polyubiquitination.

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In antiviral responses, USP21 binds to and deubiquitinates RIG-I in the cytoplasm to exert an immunomodulatory effect; USP21 can also hydrolyzes the K27/63 linked polyubiquitin chain on STING to negatively regulate DNA virus induced type I interferon production [XREF_BIBR].
USP25 deubiquitinates TRAF5. 7 / 7
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USP25 also deubiquitinated TRAF5 and TRAF6 to regulate in IL-17 signaling [XREF_BIBR].

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Taken together, our results demonstrate that USP25 negatively regulates IL-17-triggered ubiquitination of TRAF6 and TRAF5.

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Therefore, USP25 may indirectly lead to deubiquitination of TRAF5 or TRAF6 through its tightly associated proteins.

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USP25 also deubiquitinated TRAF5 and TRAF6 to regulate in IL-17 signaling [23].

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Mechanistically, stimulation with IL-17 induced the association of USP25 with the adaptors TRAF5 and TRAF6, and USP25 induced removal of Lys63-linked ubiquitination in TRAF5 and TRAF6 mediated by the adaptor Act1.

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Similarly, Usp25 deubiquitinates both TRAF5 and TRAF6 and thereby restricts downstream gene expression.

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We found that Act1 mediated K63 linked ubiquitination of TRAF5 (XREF_SUPPLEMENTARY), which was substantially attenuated by USP25 but not USP25 (C178S) in 293T cells or in an in vitro deubiquitination system (XREF_FIG and XREF_SUPPLEMENTARY).
USP25 deubiquitinates RIPK1. 7 / 7
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USP21 interacts with RIP1 and deubiquitinates RIP1 in a DUB dependent manner.

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Ubv.21.4 CDelta2 coimmunoprecipitated with USP21 in cotrans fected human embryonic kidney (HEK) 293T cells (XREF_FIG), blocked the deubiquitination of RIP1 by USP21 (XREF_FIG), and restored NF-kappaB activation (XREF_FIG).

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For example, Junichiro et al. demonstrated that USP21 constitutively deubiquitinates RIP1 in vitro and in vivo [40]; and a previous study has reported that USP20 deubiquitinates TRAF6 and Tax in vivo [40].

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USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo.

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A previous study showed that USP21 deubiquitylates receptor interacting protein 1, a suppressor of TNF induced NF-KB activation.

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Ubv.21.4 blocked the deubiquitination of RIP1 by USP21 and restored NF‐κB activation, showing that it acts as an inhibitor of USP21 in cells.

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Ubv.21.4 blocked the deubiquitination of RIP1 by USP21 and restored NF-jB activation, showing that it acts as an inhibitor of USP21 in cells.
Modified USP25 leads to the deubiquitination of DDX58. 5 / 5
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We observed that overexpression of USP25 significantly inhibited ubiquitination of RIG-I (XREF_FIG), TRAF2 (XREF_FIG), and TRAF6 (XREF_FIG).

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Overexpression of USP21 inhibited RNA virus induced RIG-I polyubiquitination and RIG-I-mediated interferon (IFN) signaling, whereas deletion of USP21 resulted in elevated RIG-I polyubiquitination, IRF3 phosphorylation, IFN-alpha and beta production, and antiviral responses in MEFs in response to RNA virus infection.

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We observed that overexpression of USP25 significantly inhibited ubiquitination of RIG-I (Figure Figure 7E ).

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We further examined the role of USP21 in the inhibition of SeV induced RIG-I polyubiquitination and found that overexpression of USP21 WT but not C221A mutant inhibited SeV induced polyubiquitination of both FLAG tagged and endogenous RIG-I (XREF_FIG).

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We observed that overexpression of USP25 significantly inhibited ubiquitination of RIG-I (Figure 7B), TRAF2 (Figure 7D), and TRAF6 (Figure 7E).
USP25 deubiquitinates TRAF3. 5 / 5
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We mentioned in a previous section that USP25 deubiquitinates TRAF3 and interacts with TRAF6, increasing expression of Tnf in HEK293T cells [XREF_BIBR, XREF_BIBR], while in MEF cells, USP25 negatively affects TNF-alpha-induced NF-kappaB activation [XREF_BIBR].

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Taking it one step further, Lin et al. uncovered that USP25 stabilizes TRAF3 in a DUB activity dependent manner by deubiquitinating K48-Ub of TRAF3 in bone marrow-derived macrophages (BMDM) cells, inhibiting TLR4 signaling-induced innate immune responses [21].

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Ubiquitin-specific protease 25 regulates TLR4-dependent innate immune responses through deubiquitination of the adaptor protein TRAF3

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We mentioned in a previous section that USP25 deubiquitinates TRAF3 and interacts with TRAF6, increasing expression of Tnf in HEK293 T cells [21,22], while in MEF cells, USP25 negatively affects TNF-α-induced NF-κB activation [20].

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Because the activation of TLR4 results in the K 48 -linked ubiquitination and degradation of TRAF3, we next determined the effects of a deficiency in USP25 on the LPS induced ubiquitination and degradation of TRAF3.
USP25 deubiquitinates FOXM1. 4 / 4
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HDACi treatment increased the ubiquitination level of FOXM1 by suppressing ubiquitin specific peptidase 21 (USP21), which deubiquitinates FOXM1.

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FOXM1 Deubiquitination by USP21 Regulates Cell Cycle Progression and Paclitaxel Sensitivity in Basal like Breast Cancer.

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Similarly, USP21 WT, but not USP21 C221A, immunopurified from 293T cells reduced polyubiquitination of FOXM1 isolated from proteasome inhibitor treated 293T cells in an in vitro deubiquitination assay (XREF_SUPPLEMENTARY).

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USP21 increases the stability of FOXM1, and USP21 binds and deubiquitinates FOXM1 in vivo and in vitro, indicating a direct enzyme-substrate relationship.
USP25 deubiquitinates GATA3. 4 / 4
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Recent reports demonstrate that GATA3 can be deubiquitinated by Usp21 which rescues it from proteosomal degradation and stabilises GATA3 protein levels [XREF_BIBR].

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Furthermore, USP21 also deubiquitinates GATA3, a transcription factor which limit T reg induced pro inflammatory responses, and its expression was noted to be upregulated in asthmatic patients.

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For example, USP21 can mediate deubiquitination of GATA3 and maintain GATA3 expression in regulatory T cells.

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Another DUB, USP21, which is itself activation induced, interacts with and deubiquitinates GATA3 to stabilize its expression in cell lines and primary human Tregs.
USP25 deubiquitinates BRCA2. 4 / 4
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To determine whether USP21 is sufficient to deubiquitinate BRCA2 fragments, we performed in vitro deubiquitination assays using HA ubiquitinated, immuno purified BRCA2 fragments as well as full-length Flag-BRCA2.

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To determine whether USP21 can promote the deubiquitination of endogenous BRCA2 and/or the BRCA2 associated RAD51 and PALB2 proteins XREF_BIBR, XREF_BIBR, we measured the extent of (poly-) ubiquitin modifications on either protein in the presence or absence of USP21 overexpression.

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We now show that USP21 interacts with and deubiquitinates BRCA2 and that USP21 loss results in decreased BRCA2 expression in tumor cell lines.

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USP21 interacts with and deubiquitinates BRCA2.
USP25 deubiquitinates MAP2K2. 4 / 4
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Since USP21 functions as a deubiquitylase, we next ask whether USP21 deubiquitylates MEK2.

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Conversely, USP21 inhibition increased MEK2 ubiquitylation in cells.

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XREF_FIG, wild type USP21 decreased MEK2 ubiquitination, whereas the C221A USP21 deubiquitinase deficient mutant failed to do so.

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USP21 deubiquitylates MEK2.
USP25 deubiquitinates EZH2. 3 / 3
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As EZH2 has been reported to promote cell metastasis in BC, our work identified that USP21 deubiquitinated EZH2 and stabilized it.

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45 USP21 could suppress EZH2 ubiquitination and thus promotes cell proliferation and metastasis in bladder carcinoma.

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Ubiquitin specific protease (USP21) deubiquitinates EZH2 and stabilizes it.
USP25 deubiquitinates TRAF2. 3 / 3
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USP25 deubiquitinates RIG-I, TRAF2 and TRAF6.

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Mechanistically, USP25 deubiquitinated retinoic acid inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor mediated IFN signaling.

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In addition, wild-type USP25 significantly inhibits ubiquitination of RIG-I, TRAF2, and TRAF6, which is essential for activation of type I IFN signaling.
USP25 deubiquitinates IFIH1. 3 / 3
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USP21 also deubiquitinated MDA5 to inhibit antiviral response [24].

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USP21 also deubiquitinated MDA5 to inhibit antiviral response [XREF_BIBR].

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Furthermore, we also found that USP21 binds to two other RLR family members (MDA5 and LGP2) and deubiquitinates MDA5, suggesting USP21 acts as a major negative regulator to restrict RLR mediated antiviral responses through deubiquitinating RIG-I and MDA5.
USP25 deubiquitinates PTEN. 2 / 2
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Conclusions: In this study, we investigated that USP25 can regulate the PI3K/AKT signaling pathway by deubiquitinating PTEN, thus affecting the proliferation and apoptosis of GCs and contributing to the pathogenesis of PCOS.

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Finally, we verified that USP25 could deubiquitinate PTEN in KGN cells.
Modified USP25 leads to the deubiquitination of PPIP5K1. 2 / 2
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However, overexpression of USP25 did not block ubiquitination of IPS-1 (Figure 7C).

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However, overexpression of USP25 did not block ubiquitination of IPS-1 (XREF_FIG).
Modified USP25 leads to the deubiquitination of TRAF2. 2 / 2
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We observed that overexpression of USP25 significantly inhibited ubiquitination of RIG-I (Figure 7B), TRAF2 (Figure 7D), and TRAF6 (Figure 7E).

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We observed that overexpression of USP25 significantly inhibited ubiquitination of RIG-I (XREF_FIG), TRAF2 (XREF_FIG), and TRAF6 (XREF_FIG).
USP25 deubiquitinates TNF. 2 / 2
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For example, USP21 deubiquitinates receptor interacting protein kinase 1 (RIP1) and repress the signaling transduction activity downstream of TNFalpha receptor 1 (TNFR1) and NFkappaB pathway [XREF_BIBR].

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Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling.
USP25 deubiquitinates SLC2A4. 2 / 2
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USP25, recruited through interaction with TNKS, could then deubiquitinate GLUT4, trapping it in GSVs and preventing its trafficking to the lysosome.

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USP25, thus positioned, could then serve to deubiquitinate GLUT4, rescuing the transporter from lysosomal degradation.
Modified USP25 leads to the deubiquitination of TRAF6. 2 / 2
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We observed that overexpression of USP25 significantly inhibited ubiquitination of RIG-I (XREF_FIG), TRAF2 (XREF_FIG), and TRAF6 (XREF_FIG).

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We observed that overexpression of USP25 significantly inhibited ubiquitination of RIG-I (Figure 7B), TRAF2 (Figure 7D), and TRAF6 (Figure 7E).
Modified USP25 leads to the deubiquitination of STING1. 2 / 2
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Our data showed that overexpression of USP21-WT, but not USP21-CA, greatly attenuated STING ubiquitination (XREF_FIG).

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Moreover, overexpression of USP21 also inhibited the HSV-1-induced STING ubiquitination (XREF_FIG).
USP25 deubiquitinates FOXP3. 2 / 2
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USP21 deubiquitinates FOXP3 in Treg cells.

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In previous studies, we showed that Foxp3 could be ubiquitinated and degraded by the E3 ubiquitin ligase Stub1 (STIP1 homology and U-Box containing protein 1) or deubiquitinated and stabilized by the deubiquitinase USP21 (ubiquitin specific peptidase 21) (Chen et al., 2013; Yang et al., 2015; Li et al., 2016).
Modified USP25 leads to the deubiquitination of TRAF3. 1 / 1
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USP25 expression indeed decreased TRAF3 polyubiquitination (XREF_FIG, lane 3).
USP25 deubiquitinates TNKS. 1 / 1
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USP25 regulates Wnt signaling by controlling the stability of tankyrases
Modified USP25 leads to the deubiquitination of FLAG. 1 / 1
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We further examined the role of USP21 in the inhibition of SeV induced RIG-I polyubiquitination and found that overexpression of USP21 WT but not C221A mutant inhibited SeV induced polyubiquitination of both FLAG tagged and endogenous RIG-I (XREF_FIG).
USP25 leads to the deubiquitination of GS26575. 1 / 1
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18, 19 USP21 can modulate cell cycle progression by deubiquitinating Forkhead box M1 (FOXM1) in basal like breast cancer.
USP25 deubiquitinates CCT4. 1 / 1
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USP25 catalyzed deubiquitination of the TRiC protein and stabilized the chaperonin, thereby reducing accumulation of misfolded polyglutamine protein aggregates.
USP25 deubiquitinates PRKDC. 1 / 1
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Mechanistically, USP21 deubiquitinated DNA-PKcs and ACLY, which led to AMPK inhibition.
USP25-C178S leads to the deubiquitination of TRAF6. 1 / 1
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Act1 mediated K63 linked ubiquitination of TRAF6 was inhibited by overexpression of USP25, but not USP25 (C178S) in 293T cells or in an in vitro deubiquitination system (XREF_FIG and XREF_SUPPLEMENTARY).
USP25 deubiquitinates BAZ2A. 1 / 1
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TIP5 is deubiquitylated and stabilized by USP21, resulting in an increase of H3K4me3 and rDNA promoter methylation.
USP25 deubiquitinates CUL. 1 / 1
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Alternatively, USP21 could be acting directly to deubiquitylate the substrates of the cullin RING ligase, in an analogous fashion to USP28, which associates with the cullin-1-adapter FBW7 to deubiquitylate a range of substrates, including MYC.
USP25 deubiquitinates retinoic acid. 1 / 1
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Mechanistically, USP25 deubiquitinated retinoic acid-inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor-associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor-mediated IFN signaling.
USP25 leads to the deubiquitination of FOSL1. 1 / 1
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These results demonstrate that USP21 enhances Fra-1 stability and AP-1 target gene expression by deubiquitinating Fra-1.
USP25 deubiquitinates IL33. 1 / 1
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Deubiquitination and stabilization of IL-33 by USP21.
USP25 deubiquitinates MARK1. 1 / 1
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USP21 deubiquitylates MARK proteins to control their stability.
USP25 leads to the deubiquitination of Forkhead. 1 / 1
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18, 19 USP21 can modulate cell cycle progression by deubiquitinating Forkhead box M1 (FOXM1) in basal like breast cancer.
USP25 deubiquitinates STING1. 1 / 1
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USP21 deubiquitinates K27/63 linked polyubiquitination of STING.
USP25 leads to the deubiquitination of SYVN1. 1 / 1
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Instead, we observed that USP25 rescued ERAD substrates and reduced levels of ubiquitination of HRD1 associated species.
USP25 deubiquitinates FOXP3 on lysine. 1 / 1
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His ubiquitin pulldown assays further confirmed that USP21 could deubiquitinate FOXP3 at these seven lysine residues and that the 7R construct was unresponsive to USP21 mediated deubiquitination (XREF_SUPPLEMENTARY).
USP25 deubiquitinates Histone. 1 / 1
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If that is the case, the global regulation of histone deubiquitination by USP21 requires further investigation.
USP25 deubiquitinates RNF135. 1 / 1
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Moreover, USP21 deubiquitinated RNF135 mediated RIG-I polyubiquitination both in vivo and in vitro (XREF_FIG).
USP25 deubiquitinates NFkappaB. 1 / 1
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For example, USP21 deubiquitinates receptor interacting protein kinase 1 (RIP1) and repress the signaling transduction activity downstream of TNFalpha receptor 1 (TNFR1) and NFkappaB pathway [XREF_BIBR].
USP25 deubiquitinates MARVELD2. 1 / 1
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USP25 catalyzed deubiquitination of the TRiC protein and stabilized the chaperonin, thereby reducing accumulation of misfolded polyglutamine protein aggregates. 0.75
Modified USP25 leads to the deubiquitination of Interferon. 1 / 1
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Overexpression of USP21 inhibited RNA virus induced RIG-I polyubiquitination and RIG-I-mediated interferon (IFN) signaling, whereas deletion of USP21 resulted in elevated RIG-I polyubiquitination, IRF3 phosphorylation, IFN-alpha and beta production, and antiviral responses in MEFs in response to RNA virus infection.
USP25 deubiquitinates isotretinoin. 1 / 1
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Mechanistically, USP25 deubiquitinated retinoic acid inducible gene I (RIG-I), tumornecrosis factor (TNF) receptor associated factor 2 (TRAF2), and TRAF6 to inhibit RIG-I-like receptor mediated IFN signaling.
USP25 deubiquitinates APP. 1 / 1
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Similarly to what occurs with?CD3delta, overexpression of?USP25?increases APP half-life. Again, this effect depends on the catalytic activity of?USP25, as catalytically inactive?USP25(C178S) does not alter APP turnover.
USP25 deubiquitinates ACLY. 1 / 1
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Mechanistically, USP21 deubiquitinated DNA-PKcs and ACLY, which led to AMPK inhibition.
USP25 deubiquitinates CD3D. 1 / 1
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Thus, the ability of USP25 to both bind and cleave ubiquitin appears to be required to exert the rescue effect on CD3未.
USP25 deubiquitinates CD274. 1 / 1
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Deubiquitination and Stabilization of PD-L1 by USP21.
USP25 leads to the deubiquitination of sensor. 1 / 1
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USP21 was also reported to antagonize the ubiquitination of anti-viral sensor RIG-I, implicating USP21 as a negative regulator of type-I interferon production and anti-viral immunity (Fan et al., 2014[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP25 deubiquitinates TCF7. 1 / 1
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Mechanistically, USP21 deubiquitinates and stabilizes the TCF/LEF transcription factor TCF7, which promotes cancer cell stemness.

Other Statements

psp cbn pc bel_lc signor biogrid lincs_drug tas hprd trrust ctd vhn pe drugbank omnipath conib crog dgi | rlimsp isi tees geneways eidos trips medscan sparser reach

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The expression of USP21 may promote proliferation, migration and invasion of breast cancer cells.

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In bladder cancer, USP21 is highly expressed and patients with high expression levels have poor survival, and USP21 can accelerate the proliferation and metastasis of bladder cancer cells via inhibiting EZH2 ubiquitination.

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USP21 promoted NSCLC cell proliferation, migration, and invasion and in vivo tumor growth by stabilizing a well-known oncogene, Yin Yang-1 (YY1), via mediating its deubiquitination.

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Therefore, USP21 stimulates cell proliferation but can not cause cell death.

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These data demonstrate that USP21 promotes cell proliferation via its deubiquitinating active site.

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USP21 promotes cell proliferation and metastasis through suppressing EZH2 ubiquitination in bladder carcinoma.

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USP21 Promotes the Proliferation, Migration, Invasion, and Stemness of GC Cells.

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In non small cell lung cancer, USP21 promotes tumor cell proliferation, migration, and invasion through the YY1 and SNHG16 axis.

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As shown in XREF_FIG, ectopic expression of USP21 obviously increased the proliferation rates of 5637 and T24 cells, whereas the inhibition of USP21 expression significantly inhibited cell proliferation.

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We found that overexpression of USP21 dramatically increased cell proliferation, while USP21-CA could not.

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In addition, ectopic, doxycyclineinducible expression of FOXM1b in MDA-MB-231 cells partially rescued the impaired proliferation caused by USP21 depletion.

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As a result, siRNA mediated depletion of USP21 inhibits cell proliferation, invasion ability and decreases the CSCs percentage of RCC cell lines.

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USP21 is upregulated in renal cell carcinoma tissues and cell lines, and depletion of USP21 inhibits cell proliferation and invasion through binding to the IL-8 promoter region and mediating transcriptional initiation 22.

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Knockdown of USP21 reduced the proliferation of MDA-MB-157 and MDA-MB-231 cells 5 days after siRNA transfection (XREF_FIG).

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Knockdown of USP21 in TNBC cells inhibited cell proliferation, migration and invasion.

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Several functional experiments, including colony formation analysis and CCK-8 analysis, suggested that overexpression of USP21 promoted cell proliferation and inhibition of USP21 suppressed cell proliferation.

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USP21 knockdown or overexpression in the DLBCL cell line shows that USP21 promotes cell proliferation.

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Accordingly, BCR-ABL-mediated signaling and cell proliferation were suppressed in BCR-ABL-positive leukemia cells by the depletion of USP25.

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Therefore, while depletion of USP21 (and FOXM1) impairs proliferation in each of these cell lines under control conditions, there is a statistically significant decrease in viability when USP21 depletion is combined with paclitaxel treatment.

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While cell proliferation rates were comparable at early time points examined, we found that USP21 depletion led to dramatically decrease of cell proliferation 6 days after transfection.
USP25 affects IFNB1
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USP25 inhibits IFNB1.
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(F) Effects of USP25 siRNA on SEV-induced activation of the IFN-β promoter.

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Both USP21 and A20 inhibited SeV- and RIG-I-CARD-induced IFN-beta reporter activity in a dose dependent manner.

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Fan et al., knowing that USP21 inhibits RIG-I-induced IFN-beta production, searched for its mechanism [XREF_BIBR].

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Interestingly, we found that overexpression of USP21 WT but not C221A mutant inhibited IFN-beta, NF-kappaB, and ISRE reporter activities in HEK293T cells in response to transfected poly (I : C), which was known to be mediated by MDA5 (unpublished data), suggesting that USP21 might also target MDA5.

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Cellular ubiquitin specific proteases, USP21, USP3 and USP15, a subfamily of deubiqutinase, remove K63 linked polyubiquitin chains from RIG-I and block it to induce IFN-beta.

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The results showed that USP25-WT remarkably inhibited SEV-induced activation of IFN-β (Figure 6A), IRF3 (Figure 6B), NF-κB (Figure 6C) and ISRE (Figure 6D) in a dose-dependent manner.

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The results showed that USP25 significantly reduced SEV-induced IFN-β luciferase reporter activity (Figure 2A).

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Reporter assays then indicated that knockdown of USP25 markedly potentiated SEV induced activation of the IFN-beta promoter (XREF_FIG).

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The results showed that USP25-WT remarkably inhibited SEV-induced activation of IFN-β ( Figure 6A ), IRF3 ( Figure 6B ), NF-κB ( Figure 6C ) and ISRE ( Figure 6D ) in a dose-dependent To further determine the levels at which USP25 negatively regulates type I IFN signaling, HEK-293T cells were transfected with DNA constructs encoding RIG-I, IPS-1, TRAF2, or TRAF6, together with IFN-β-Luc.

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Overexpression of RNF135 alone could not induce IFN-beta reporter activity, whereas once co-overexpressed with RIG-I, RNF135 robustly induced IFN-beta reporter activity, which was strongly inhibited by USP21 WT but not C222A mutant (XREF_FIG and not depicted).
USP25 bound to IFIH1 inhibits IFNB1. 1 / 1
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Overexpression studies in HEK293T cells revealed that USP21 might also bind and deubiquitinate MDA5 by removing K63 linked polyubiquitination, thus inhibiting IFN-beta, NF-kappaB, and IFN stimulated response element (ISRE) reporter activities.
USP25 decreases the amount of IFNB1.
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USP25 decreases the amount of IFNB1. 4 / 4
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Our data showed that Usp21 knockdown significantly enhanced Ifnb expression in WT but not in Sting -/- MEFs (XREF_FIG).

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Taken together, our results suggest that USP25 negatively regulates IFN-β expression by inhibiting SEV-induced activation of IRF3 and NF-κB.USPs are cysteine proteases that vary greatly in size and structural complexity.

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Taken together, our results suggest that USP25 negatively regulates IFN-beta expression by inhibiting SEV induced activation of IRF3 and NF-kappaB.

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We found that knockdown of Usp21 in mouse fibroblast L929 cells significantly enhanced the expression of Ifnb, Ifna4, and Isg15 (XREF_FIG).
Modified USP25 decreases the amount of IFNB1. 1 / 1
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The overexpression of USP25 downregulated IFN-beta promoter activity and IFN-beta expression in SeV infected HEK293T cells, and the silencing of USP25 by siRNA enhanced the IFN-beta promoter activity [XREF_BIBR].
USP25 activates IFNB1.
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USP25 activates IFNB1. 4 / 4
| 1 3

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The results showed that USP25-WT remarkably inhibited SEV-induced activation of IFN-β ( Figure 6A ), IRF3 ( Figure 6B ), NF-κB ( Figure 6C ) and ISRE ( Figure 6D ) in a dose-dependent To further determine the levels at which USP25 negatively regulates type I IFN signaling, HEK-293T cells were transfected with DNA constructs encoding RIG-I, IPS-1, TRAF2, or TRAF6, together with IFN-β-Luc.

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Notably, Usp21 fl/fl Lyz2-cre mice produced significantly higher concentrations of IFNbeta, IL-6, and TNF in serum upon HSV-1 infection compared with infected control mice (XREF_FIG).

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Furthermore, Knockdown of USP25 potentiated virus induced induction of the IFN-beta.

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Consistently, Usp21 deficiency resulted in production of more IFNbeta and TNF in MEFs and BMDMs in response to HSV-1 infection (XREF_FIG).
USP25 increases the amount of IFNB1.
| 2
USP25 increases the amount of IFNB1. 2 / 2
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Usp21 ablation also drastically enhanced the expression of Ifnb, Ifna4, or TNF mRNA in primary PEMs (peritoneal macrophages; XREF_FIG) or BMDMs (BM derived macrophages; XREF_FIG).

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USP25 's DUB activity was also found to be necessary for virus induced signaling, as USP25 knockdown MEFs with WT USP25 reconstitution allowed expression of Ifnb, Ifna4 and IL-6 upon SeV or HSV-1 induction, while those with DUB activity mutant USP25 did not [XREF_BIBR].
USP25 affects Interferon
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USP25 inhibits Interferon.
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However, the catalytic mutants (C178A and H607A) devoid of DUB activity lost the ability of USP25 WT-mediated IFN inhibition to some degree, indicating that DUB activity is involved in USP25 inhibition of type I IFN induction.To further determine the levels at which USP25 negatively regulates type I IFN signaling, HEK-293T cells were transfected with DNA constructs encoding RIG-I, IPS-1, TRAF2, or TRAF6, together with IFN-β-Luc.

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We further investigated the effect of USP25 knockdown on virus triggered IFN signaling.

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However, the catalytic mutants (C178A and H607A) devoid of DUB activity lost the ability of USP25 WT mediated IFN inhibition to some degree, indicating that DUB activity is involved in USP25 inhibition of type I IFN induction.

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In addition, the catalytic USP25 mutants (C178A and H607A) devoid of DUB activity significantly lost the ability of USP25 WT mediated IFN inhibition.

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To examine whether the inhibitory effects of USP25 on SEV-induced type I IFN signaling is due to its deubiquitinase activity, wild-type USP25 (USP25-WT) and its mutants (C178A and H607A) lacking DUB activity were co-transfected with the promoter luciferase reporter plasmid of IFN-β, IRF3, NF-κB and ISRE, and the luciferase activity was detected.

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USP25 inhibits SEV induced type I IFN signaling by disrupting activation of IRF3 and NF-kappaB.

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The input tagged proteins were verified with the indicated antibodies.doi: 10.1371/journal.pone.0080976.g002 USP25 Negatively Regulates Type I IFN Signaling PLOS ONE | www.plosone.orgdoi: 10.1371/journal.pone.0080976.g003 USP25 Negatively Regulates Type I IFN Signaling PLOS ONE | www.plosone.org manner.

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Collectively, these findings suggest that USP25 inhibits RIG-I and MDA5-dependent type I IFN signaling.

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However, the catalytic mutants (C178A and H607A) devoid of DUB activity lost the ability of USP25 WT-mediated IFN inhibition to some degree, indicating that DUB activity is involved in USP25 inhibition of type I IFN induction.Sequence for siRNAs of human USP25.

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Interestingly, the genetic deletion of USP21 in macrophages enhances IRF3 activation, IFN production, and antiviral response.
USP25 bound to IFIH1 inhibits Interferon. 1 / 1
| 1

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Overexpression studies in HEK293T cells revealed that USP21 might also bind and deubiquitinate MDA5 by removing K63 linked polyubiquitination, thus inhibiting IFN-beta, NF-kappaB, and IFN stimulated response element (ISRE) reporter activities.
USP25 activates Interferon.
| 1 5
| 1 5

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Intraperitoneal injection of VSV in 4-wk-old USP21 -/- also induced a stronger IFN production compared with that in 4-wk-old WT mice (unpublished data).

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Mutation of the catalytic residues results in significant loss of ability of USP25 mediated IFN inhibition.

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However, our studies explain that USP25 negatively modulates RIG-I/MDA5-dependent type I IFN signaling.

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In addition , USP25 deficiency inhibits transcriptional activity of interferon regulatory factor , thereby reducing type I interferon production ( 20 ) .
| PMC

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However, our studies explain that USP25 negatively modulates RIG-I and MDA5-dependent type I IFN signaling.

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USP25 restricts proinflammatory cytokine production and promotes type I IFN production upon LPS stimulation.
USP25 increases the amount of Interferon.
| 1
USP25 increases the amount of Interferon. 1 / 1
| 1

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Several other DUBs have also been implicated in the regulation of RIG-I ubiquitination and virus induced type I IFN expression; these include A20, USP3, USP15, USP21, and USP25 XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR.
USP25 affects DDX58
| 17
USP25 inhibits DDX58.
| 12
| 12

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Collectively, these findings suggest that USP25 inhibits RIG-I and MDA5-dependent type I IFN signaling.

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In particular, USP3, USP21, USP25 and USP15 have all been shown to directly inhibit RIG-I.

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The deubiquitinases USP3 and USP21 inhibit RIG-I activity by removing K63-linked ubiquitin chains.
| PMC

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RIG-I is negatively regulated by a deubiquitinase, USP21 .
| PMC

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USP21 and USP15 remove K63-linked polyubiquitin chains from RIG-I and block the ability of RIG-I to induce IFN-β 24,25 .

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USP3, USP21 and CYLD inhibit RIG-I K63-linked ubiquitination and activation.

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RIG-I is negatively regulated by a deubiquitinase, USP21 (Fan et al. 2014).
| PMC

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Similarly, USP21 has been reported to negatively regulate RIG-I by removing K63 linked ubiquitination [XREF_BIBR].

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Conversely, the removal of Lys63 linked ubiquitylation by the cellular deubiquitylating enzymes (DUBs) ubiquitin C-terminal hydrolase 3 (USP3), USP21 and CYLD, represses RIG-I signalling.

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Interestingly, USP21 inhibits both TRIM25- and RNF135 mediated antiviral response and RIG-I activation (XREF_FIG and not depicted).
USP25 activates DDX58.
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USP25 activates DDX58. 5 / 5
| 5

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However, our studies explain that USP25 negatively modulates RIG-I/MDA5-dependent type I IFN signaling.

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USP17 and USP25 also deubiquitinate RIG-I in a different manner; that is, USP17 stabilizes RIG-I by K48-deubiquitination, while USP25 inhibits RIG-I degradation by K63-deubiquitination [37, 38] .

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The deubiquitinase USP21 targets RIG-I for deubiquitination, thus dampening interferon production and activation of IFN responsive genes in response to RNA viruses.

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However, our studies explain that USP25 negatively modulates RIG-I and MDA5-dependent type I IFN signaling.

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Ubiquitylation events are reversible processes, and, accordingly, several deubiquitylating enzymes, in particular ubiquitin specific peptidase 3 (USP3), USP21 and CYLD lysine 63 deubiquitinase (CYLD), modulate RIG-I signalling by removing K63-polyubiquitin chains, although with unique kinetics.
| 13

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USP21 Promotes the Proliferation, Migration, Invasion, and Stemness of GC Cells.

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Afterward, the role of USP21 in cell migration and invasion was evaluated through Transwell assay, and the data in XREF_FIG showed that overexpression of USP21 accelerated the migration and invasion of AGS cells, whereas knockdown of USP21 pronouncedly reduced the migration and invasion of MKN-45 cells.

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These results demonstrated that USP21 stimulated cell proliferation, migration, invasion, and stemness of GC cells.

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The expression of USP21 may promote proliferation, migration and invasion of breast cancer cells.

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In non small cell lung cancer, USP21 promotes tumor cell proliferation, migration, and invasion through the YY1 and SNHG16 axis.

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USP21 promotes migration and invasion ability of BC cells and facilitates EMT.

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Overexpression of USP21 increase the cell growth, invasion and cancer stem cell percentage of 786-O and A-704 cells.

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Thus far, a study proved that USP21 can accelerate cell growth, invasion, and stemness of renal cell carcinoma.

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USP21 promoted NSCLC cell proliferation, migration, and invasion and in vivo tumor growth by stabilizing a well-known oncogene, Yin Yang-1 (YY1), via mediating its deubiquitination.

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XREF_BIBR, XREF_BIBR Recently, one report indicated that USP21 promotes cell proliferation and invasion ability in human renal cell carcinoma.

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USP21 is upregulated in renal cell carcinoma tissues and cell lines, and depletion of USP21 inhibits cell proliferation and invasion through binding to the IL-8 promoter region and mediating transcriptional initiation 22.

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Knockdown of USP25 by siRNA in A549, H1299, and SPC-A-1sci cells inhibited cell migration and invasion in vitro, which fell to levels similar to those observed after transfection with the miR-200c mimics.

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Knockdown of USP21 in TNBC cells inhibited cell proliferation, migration and invasion.

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Knockdown of USP21 decreased the cell growth, invasion and cancer stem cell percentage of 786-O cells.

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Knockdown of USP21 decreased the cell growth, invasion and cancer stem cell percentage of A-704 cells.
USP25 affects NFkappaB
| 3 12
USP25 inhibits NFkappaB.
| 11
| 10

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The results showed that USP25-WT remarkably inhibited SEV induced activation of IFN-beta (XREF_FIG), IRF3 (XREF_FIG), NF-kappaB (XREF_FIG) and ISRE (XREF_FIG) in a dose dependent manner.

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Because USP25 negatively regulates IL-17-induced activation of NF-kappaB and stabilization of chemokine mRNA, which involves TRAF6 and TRAF5, respectively 26, we examined whether USP25 interacts with these TRAF proteins.

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The deubiquitinases (DUBs) A20 and USP25 negatively regulate IL-17-induced NF-kappaB and MAPK activation by removing ubiquitin modifications on TRAF6.

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USP21 was reported to target RIPK1 and inhibit NF-kappaB activity 96, however mice lacking USP21 show no defect in NF-kappaB signalling 93.

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Interestingly, we found that overexpression of USP21 WT but not C221A mutant inhibited IFN-beta, NF-kappaB, and ISRE reporter activities in HEK293T cells in response to transfected poly (I : C), which was known to be mediated by MDA5 (unpublished data), suggesting that USP21 might also target MDA5.

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We observed that overexpression of USP25 significantly inhibited SEV induced activation of IRF3 and NF-kappaB (XREF_FIG).

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The analysis suggested that reconstitution of USP25 almost completely inhibited IL-17-induced activation of NF-kappaB compared to USP25 (C178S) (0.89 v.s 1.8 at 15 min, 0.61 v.s. 8.3 at 30 min, respectively) (XREF_FIG).

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Because IFN-beta reporter activity is dependent on both NF-kappaB and IRF3, we further tested the inhibitory effect of USP21 on SeV-, RIG-I-CARD-, and TBK1 induced NF-kappaB and ISRE reporter activities.

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To better understand the effects of USP25 in SEV induced type I IFN signaling, we assessed whether expression of USP25 disrupts SEV induced activation of IRF3 and NF-kappaB, the two important transcriptional factors in type I IFN signaling.

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USP21 inhibits NF-kB activation through the deubiquitylation of RIPK1 [XREF_BIBR].
USP25 bound to IFIH1 inhibits NFkappaB. 1 / 1
| 1

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Overexpression studies in HEK293T cells revealed that USP21 might also bind and deubiquitinate MDA5 by removing K63 linked polyubiquitination, thus inhibiting IFN-beta, NF-kappaB, and IFN stimulated response element (ISRE) reporter activities.
USP25 activates NFkappaB.
| 3 1
| 3 1

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An additional study has revealed that depletion of USP21 decreases IL33 protein levels and IL33 mediated NF-kappaB p65 promoter activity, indicating USP21 is able to positively regulate the NF-kappaB signaling pathway.

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We found that the DUB, USP25 xref inhibited IL-17-but not TNF-induced activation of NF-κB in reporter assays in HeLa cells and in 293T cells transfected with IL-17RA and IL-17RC (293T-IL-17RA/C) ( xref and xref ).

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The results showed that USP25-WT remarkably inhibited SEV-induced activation of IFN-β ( Figure 6A ), IRF3 ( Figure 6B ), NF-κB ( Figure 6C ) and ISRE ( Figure 6D ) in a dose-dependent To further determine the levels at which USP25 negatively regulates type I IFN signaling, HEK-293T cells were transfected with DNA constructs encoding RIG-I, IPS-1, TRAF2, or TRAF6, together with IFN-β-Luc.

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The analysis suggested that reconstitution of USP25 almost completely inhibited IL-17-induced activation of NF-κB compared to USP25(C178S) (0.89 v.
USP25 affects IRF3
| 1 11
USP25 inhibits IRF3.
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USP25 inhibits IRF3. 10 / 11
| 10

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We observed that overexpression of USP25 significantly inhibited SEV induced activation of IRF3 and NF-kappaB (XREF_FIG).

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Collectively, these findings suggest that USP25 inhibits RIG-I/MDA5-dependent type I IFN signaling.To better understand the effects of USP25 in SEV-induced type I IFN signaling, we assessed whether expression of USP25 disrupts SEV-induced activation of IRF3 and NF-κB, the two important transcriptional factors in type I IFN signaling.

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USP21 inhibits RIG-I-CARD-mediated IRF3 activation and negatively regulates antiviral response.

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The results showed that USP25-WT remarkably inhibited SEV induced activation of IFN-beta (XREF_FIG), IRF3 (XREF_FIG), NF-kappaB (XREF_FIG) and ISRE (XREF_FIG) in a dose dependent manner.

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To better understand the effects of USP25 in SEV induced type I IFN signaling, we assessed whether expression of USP25 disrupts SEV induced activation of IRF3 and NF-kappaB, the two important transcriptional factors in type I IFN signaling.

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Interestingly, the genetic deletion of USP21 in macrophages enhances IRF3 activation, IFN production, and antiviral response.

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We observed that overexpression of USP25 significantly inhibited SEV-induced activation of IRF3 and NF-κB (Figure 3A and B).

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USP21 inhibits virus induced IRF3 activation via binding to and deubiquitinating RIG-I.

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The results showed that USP25-WT remarkably inhibited SEV-induced activation of IFN-β (Figure 6A), IRF3 (Figure 6B), NF-κB (Figure 6C) and ISRE (Figure 6D) in a dose-dependent manner.

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The results showed that USP25-WT remarkably inhibited SEV-induced activation of IFN-β ( Figure 6A ), IRF3 ( Figure 6B ), NF-κB ( Figure 6C ) and ISRE ( Figure 6D ) in a dose-dependent To further determine the levels at which USP25 negatively regulates type I IFN signaling, HEK-293T cells were transfected with DNA constructs encoding RIG-I, IPS-1, TRAF2, or TRAF6, together with IFN-β-Luc.
USP25 activates IRF3.
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USP25 activates IRF3. 2 / 2
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USP21 deficiency enhances intracellular IRF3 activation.

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The results showed that USP25-WT remarkably inhibited SEV-induced activation of IFN-β ( Figure 6A ), IRF3 ( Figure 6B ), NF-κB ( Figure 6C ) and ISRE ( Figure 6D ) in a dose-dependent To further determine the levels at which USP25 negatively regulates type I IFN signaling, HEK-293T cells were transfected with DNA constructs encoding RIG-I, IPS-1, TRAF2, or TRAF6, together with IFN-β-Luc.
USP25 affects FOXP3
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USP25 activates FOXP3.
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USP25 activates FOXP3. 4 / 4
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Here USP21 prevents FOXP3 degradation most likely through removing K48 linked polyubiquitin moieties.

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USP21 prevents the loss of FOXP3 protein in Treg cells.

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Therefore, USP21 prevents the proteasomal degradation of FOXP3 in Treg cells.

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Interestingly, Foxp3 directly activates expression of USP21, and siRNA knockdown of USP21 downregulates both GATA3 and Foxp3 protein.
USP25 activates FOXP3. 2 / 2
| 2

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USP21 prevents FOXP3 degradation through deubiquination, thus stabilizing Treg phenotype and antagonizing the development of Th1 like Tregs [XREF_BIBR].

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Similarly, USP21 prevents Foxp3 degradation by deubiquitinating K48-type modifications at residues Lys 206, Lys 216, Lys 227, Lys 252, Lys 277, Lys 332, and Lys 393.
USP25 inhibits FOXP3.
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USP25 inhibits FOXP3. 4 / 4
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Our results demonstrate how USP21 prevents FOXP3 protein depletion and controls Treg lineage stability in vivo.

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Therefore, these results support that USP21 prevents FOXP3 depletion in Treg cells through deubiquitination and protection from ubiquitination mediated protein degradation.

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Here we report that the E3 deubiquitinase USP21 prevents the depletion of FOXP3 at the protein level and restricts the generation of T-helper-1-like Treg cells.

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Taken together, our results show that USP21 prevents FOXP3 protein depletion and controls Treg lineage stability in vivo.
USP25 increases the amount of FOXP3.
| 3
USP25 increases the amount of FOXP3. 2 / 2
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It is likely that by stabilizing GATA3 levels, USP21 indirectly supports Foxp3 expression and Treg function.

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This last report proposed that after TCR stimulation, FOXP3 upregulates USP21 transcription.
USP25 increases the amount of FOXP3. 1 / 1
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166 Treg specific deletion of USP21 in mice reduces the level of Foxp3 and perturbs the expression of Treg signature genes, causing aberrant T cell activation and autoimmune symptoms.
USP25 affects FOXM1
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USP25 activates FOXM1.
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USP25 activates FOXM1. 8 / 8
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The expression of USP21 WT increased FOXM1 stability, and its half-life was approximately 12.4 h.

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USP21 activated the Hippo pathway by mediating FOXM1.

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This is consistent with our previous data demonstrating that FOXM1 abundance is positively enhanced by USP21 overexpression.

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USP21 overexpression significantly enhanced FOXM1 dependent transcriptional activity compared with control (XREF_FIG).

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Because FOXM1 is upregulated by USP21, we asked whether a curated list of 114 FOXM1 target genes is correlated with USP21 amplification using gene set enrichment analysis (GSEA).

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USP21 increases the stability of FOXM1, and USP21 binds and deubiquitinates FOXM1 in vivo and in vitro, indicating a direct enzyme-substrate relationship.

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USP21 stabilizes FOXM1, and suppressing USP21 reduces FOXM1 abundance, which subsequently downregulates the FOXM1 transcriptional network.

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Correspondingly, ectopic expression of USP21 significantly increased FOXM1 abundance in 293T cells (XREF_FIG).
USP25 inhibits FOXM1.
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USP25 inhibits FOXM1. 2 / 2
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Depleting USP21 downregulates the FOXM1 transcriptional network and causes a signifi-cant delay in cell cycle progression.

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Similarly, depletion of USP21 in MDA-MB-231 reduced FOXM1 half-life from 11.5 to 4.2 h (XREF_SUPPLEMENTARY).
USP25 increases the amount of FOXM1.
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USP25 increases the amount of FOXM1. 1 / 1
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IB analysis of cell lysates 48 h after transfection revealed that USP21 knockdown reproducibly reduced the level of endogenous FOXM1 (XREF_FIG).
USP25 decreases the amount of FOXM1.
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USP25 decreases the amount of FOXM1. 1 / 1
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USP21 knockdown did not lead to the same decrease in FOXM1 transcriptional target abundance as FOXM1 knockdown (XREF_FIG, compare red bars to orange bars); however, this is consistent with biochemical data demonstrating that USP21 knockdown reduces FOXM1 levels to an amount between what is observed in control conditions and what is observed in FOXM1 knockdown conditions (XREF_FIG).
USP25 affects BRCA2
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USP25 activates BRCA2.
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USP25 activates BRCA2. 7 / 7
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We next asked whether USP21 mediated BRCA2 stabilization involves USP21 interaction with the BRCA2 and RAD51 complex.

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We next asked if USP21 mediated BRCA2 fragment stabilization is associated with changes in poly-ubiquitination.

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Our data collectively point to a mechanism of USP21 mediated BRCA2 stabilization via proteasomal degradation.

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To gain mechanistic insight into USP21 function in HCC, we asked whether USP21 can modulate BRCA2 stability in HCC derived tumor cells 50.

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USP21-mediated BRCA2 stabilization promotes RAD51 loading at DSB sites.
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USP21 stabilizes BRCA2 in patient derived HCC tumor cell lines, protects from DNA damage and promotes tumor cell growth in a BRCA2 dependent manner.

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Together, these findings support the notion that USP21 promotes BRCA2 stability and protects from DNA damage accumulation in BRCA2-proficient tumor cells, which can in turn contribute to increased tumor growth and, ultimately, a more malignant phenotype.
USP25 increases the amount of BRCA2.
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USP25 increases the amount of BRCA2. 2 / 2
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USP21 knockdown with two independent shRNAs caused a reduction in BRCA2 protein levels that was particularly pronounced in the presence of the topoisomerase I inhibitor camptothecin (CPT), which causes DSB induction in HR-permissive S phase cells 36.

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USP21 loss causes a decrease in BRCA2 protein levels.
Modified USP25 increases the amount of BRCA2. 1 / 1
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Notably, USP21 overexpression caused a significant, DUB activity dependent increase in BRCA2 fragment levels that correlated with the extent of USP21 interaction and was most pronounced for the OB domain containing constructs.
USP25 inhibits BRCA2.
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USP25 inhibits BRCA2. 1 / 1
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Importantly, we provide evidence that a defect in USP21 mediated BRCA2 stabilization impairs the growth of BRCA2-proficient HCC tumor cells, and consistent with this, USP21 levels inversely correlate with HCC patient survival, pointing to USP21 as a potential target for HCC tumor therapy.
USP25 decreases the amount of BRCA2.
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USP25 decreases the amount of BRCA2. 1 / 1
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Consistent with this, inhibition of proteasome activity was able to stabilize BRCA2 and reverse the reduction in BRCA2 protein levels following USP21 depletion.
USP25 affects NANOG
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USP25 activates NANOG.
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USP25 activates NANOG. 5 / 5
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Co-expression of either USP21 isoform significantly prolonged the half-life of Nanog (XREF_FIG).

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Overexpression of USP21 prolonged the protein half-life of Nanog to ~ 2h, whereas the catalytically inactive mutant USP21 C221A had no such effects (XREF_FIG).

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They further demonstrated that USP21 prevents the degradation of Nanog through deubiquitylation and thus promote maintenance of embryonic stem cells (ESCs).

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USP21 mediated Nanog stabilization is enhanced in mouse ESCs and this stabilization is required to maintain the pluripotential state of the ESCs.

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USP21 could significantly enhance the stability of Nanog and then maintain the self-renewal of stem cells.
USP25 increases the amount of NANOG.
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USP25 increases the amount of NANOG. 3 / 3
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Through the screen, we found that USP21 significantly upregulated Nanog levels, whereas other DUBs had little to no effect on the Nanog expression levels (XREF_FIG).

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Through the screen they also found that USP21 significantly upregulated Nanog levels while other DUBs had little to no effect on the Nanog expression levels.

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Overexpression of USP21, but not the catalytically inactive mutant C221A, led to increased Nanog levels in a dose dependent way (XREF_FIG), suggesting that USP21 upregulation of Nanog expression is dependent on DUB enzyme activity.
Modified USP25 increases the amount of NANOG. 1 / 1
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Overexpression of USP21, but not the catalytically inactive mutant C221A, led to increased Nanog levels in a dose dependent way (XREF_FIG), suggesting that USP21 upregulation of Nanog expression is dependent on DUB enzyme activity.
USP25 inhibits NANOG.
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USP25 inhibits NANOG. 1 / 1
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Depletion of endogenous USP21 by short hairpin RNA (shRNA) in mESC E14 cells reduced both the protein level (XREF_FIG) and half-life of Nanog, but not the half-life of other pluripotent factors such as Sox2, Oct4 and Klf4 (XREF_FIG).
USP25 affects GATA3
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USP25 activates GATA3.
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USP25 activates GATA3. 6 / 6
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Overexpression of USP21 can rescue GATA3 from its degradation so as to stabilize the expression of GATA3 [XREF_BIBR]; RT-PCR showed that the mRNA of USP21 is upregulated in the Treg cells of asthma patients.

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USP21 positively regulates and stabilizes GATA3, which can maintain FOXP3 expression.

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In a search for DUBs that contribute to GATA3 stabilization in FOXP3-expressing cells, both USP7 and USP21 were shown to upregulate GATA3-mediated activity using a reporter assay 131 .

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Expression of USP21 leads to stabilize GATA3.

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In summary, we have identified a USP21 mediated pathway that promotes GATA3 stabilization and expression at the post-translational level.

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Interestingly, Foxp3 directly activates expression of USP21, and siRNA knockdown of USP21 downregulates both GATA3 and Foxp3 protein.
USP25 increases the amount of GATA3.
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USP25 increases the amount of GATA3. 2 / 2
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It is likely that by stabilizing GATA3 levels, USP21 indirectly supports Foxp3 expression and Treg function.

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In the terms of GATA3, E3 deubiquitinase ubiquitin specific peptidase 21 (USP21) can upregulate the expression of GATA3 in Treg cells [XREF_BIBR].
USP25 inhibits GATA3.
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USP25 inhibits GATA3. 1 / 1
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Zhang et al. have reported that in Treg cells, the DUB USP21 interacted with and stabilized GATA3 via deubiquitination with the overexpression of USP21 obviously decreasing the ubiquitination status of GATA3.
USP25 decreases the amount of GATA3.
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USP25 decreases the amount of GATA3. 1 / 1
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While simultaneous knockdown of MAPK1 and overexpression of USP21 could conspicuously increase the expression of GATA3 and MAPK1 (XREF_FIG), indicating that USP21 could regulate MAPK1 through GATA3.
USP25 affects cell growth
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USP25 activates cell growth.
| 7
| 7

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Overexpression of USP21 increase the cell growth, invasion and cancer stem cell percentage of 786-O and A-704 cells.

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We identify that knockdown of USP21 inhibits cell proliferation, and overexpression of USP21 promotes cell growth.

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USP21 stabilizes BRCA2 in patient derived HCC tumor cell lines, protects from DNA damage and promotes tumor cell growth in a BRCA2 dependent manner.

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Amplification of USP21 deubiquitinase promotes pancreatic cancer cell growth and stemness via Wnt and beta-catenin signaling [XREF_BIBR].

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Thus far, a study proved that USP21 can accelerate cell growth, invasion, and stemness of renal cell carcinoma.

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Here, we have shown that USP21 over-expression promotes HCC cell growth, cell cycle progression and in vivo tumorigenesis, while knockdown of USP21 inhibits these processes.

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USP21 deubiquitinates and stabilizes BRCA2, promotes HR efficiency, and enhances homologous recombination efficiency and tumor cell growth.
USP25 inhibits cell growth.
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Knockdown of USP21 decreased the cell growth, invasion and cancer stem cell percentage of 786-O cells.

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Knockdown of USP21 decreased the cell growth, invasion and cancer stem cell percentage of A-704 cells.
USP25 affects Ubiquitin
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USP25 activates Ubiquitin.
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Deletion of one or both UIM domains of USP25 does not alter C-terminal processing activity but strongly impairs K48 and K63 ubiquitin chain cleavage.

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To further identify which Ub linkage type is targeted by USP25 in vivo, HEK-293T cells were transfected with HA-K48-Ub or HA-K63-Ub in lieu of HA-Ub.

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Interestingly, we observed the opposite pattern with USP21, which rescued ubiquitin availability but was unable to rescue degradation of our panel of substrates.

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Additionally USP21 may target not only ubiquitin conjugated substrates but also substrates conjugated to ubiquitin like proteins ISG15 and NEDD8 (Gong et al., 2000; Ye et al., 2011).
USP25 inhibits Ubiquitin.
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Overexpression of USP21 significantly reduced ubiquitin incorporation into MARK1 but has less impact on the MARK1K768A mutant (XREF_SUPPLEMENTARY).

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USP21 inhibits antiviral response independent of A20 ubiquitin editing complex.
USP25 decreases the amount of Ubiquitin.
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USP25 decreases the amount of Ubiquitin. 2 / 2
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USP21 and Cezanne inhibit TNF-κB activation by regulating ubiquitin level of RIPK1 (D'Arcy et al., 2015).

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USP21 and Cezanne inhibit TNF-B activation by regulating ubiquitin level of RIPK1 (D'Arcy et al., 2015) .
USP25 increases the amount of Ubiquitin.
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USP25 increases the amount of Ubiquitin. 1 / 1
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As shown in Figure XREF_FIG, USP21 depletion increased the amount of ubiquitin detected on MARK1 purified by IP.
USP25 affects STING1
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USP25 inhibits STING1.
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USP25 inhibits STING1. 4 / 4
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The deubiquitinase USP21 can negatively regulate STING activity by removing K27- and K63 linked ubiquitin chains of STING [XREF_BIBR].

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Our data showed that phosphorylation of USP21 by p38 promotes its binding to STING and inactivates STING in response to HSV-1 infection.

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Hence, at late stages of viral infection, p38-mediated phosphorylation of USP21 (Ubiquitin Specific Peptidase 21), a deubiquitinating enzyme, inhibits STING.

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Our data showed that overexpression of USP21 blocked translocation of STING from ER to a perinuclear microsome upon HSV-1 infection (XREF_FIG).
Modified USP25 inhibits STING1. 1 / 1
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Ectopic expression of USP21 blocked the interaction between STING and IRF3 (XREF_FIG) and significantly reduced STING multimerization (XREF_FIG).
USP25 bound to STING1 inhibits STING1. 1 / 1
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We found that prolonged DNA virus infection induces the phosphorylation of USP21 at Ser538 by activation of p38 MAPK, which in turn promotes the binding of USP21 to STING, deubiquitinates and inactivates STING.
USP25 activates STING1.
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USP25 activates STING1. 2 / 2
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Importantly, GST pull-down assay showed that phosphorylation of USP21 by activated p38 significantly enhanced the binding of USP21 to the purified STING (XREF_FIG).

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Our data indicated that microtubule association is not required for USP21 mediated STING inactivation (unpublished data).
USP25 decreases the amount of STING1.
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USP25 decreases the amount of STING1. 1 / 1
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In this study, we found that USP21 negatively regulates STING mediated antiviral gene expression, as well as host antiviral response.
USP25 affects APP
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USP25 activates APP.
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USP25 activates APP. 4 / 4
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We investigated whether USP25 prevents APP degradation under ER-stress conditions.

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Together, these findings suggest that the activation of A23187 mediated ERAD resulted in APP degradation, and that USP25 inhibits APP degradation by the proteasome.

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Similarly to what occurs with CD3delta, overexpression of USP25 increases APP half-life.

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The positive effect of USP25 on APP protein is detectable only when the proteasome is active; treatment of transfected cells with the inhibitor MG132 abolishes this effect (XREF_FIG), suggesting that USP25 rescues APP from proteasomal degradation.
USP25 inhibits APP.
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USP25 inhibits APP. 2 / 3
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We confirmed that A23187 induced APP degradation was rescued by the overexpression of USP25 (XREF_FIG).

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In cycloheximide based time course experiments, where production of new protein is halted, USP25 modestly, but statistically significantly, slows down APP turnover (XREF_FIG).
USP25 increases the amount of APP.
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USP25 increases the amount of APP. 1 / 1
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We tested whether RNAi mediated knockdown of endogenous USP25 causes a reduction in endogenous APP protein levels.
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Valproic acid increases the amount of USP25. 7 / 7
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USP25 affects SGCG
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USP25 inhibits SGCG.
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USP25 inhibits SGCG. 6 / 6
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However, the catalytic mutants (C178A and H607A) devoid of DUB activity lost the ability of USP25 WT-mediated IFN inhibition to some degree, indicating that DUB activity is involved in USP25 inhibition of type I IFN induction.Sequence for siRNAs of human USP25.

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The input tagged proteins were verified with the indicated antibodies.doi: 10.1371/journal.pone.0080976.g002 USP25 Negatively Regulates Type I IFN Signaling PLOS ONE | www.plosone.orgdoi: 10.1371/journal.pone.0080976.g003 USP25 Negatively Regulates Type I IFN Signaling PLOS ONE | www.plosone.org manner.

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*P < 0.05 for all pairwise comparisons by one-way ANOVA followed by Dunnett's multiple comparisons test.doi: 10.1371/journal.pone.0080976.g006 USP25 Negatively Regulates Type I IFN Signaling PLOS ONE | www.plosone.orgUSP25 deubiquitinates RIG-I, TRAF2 and TRAF6.

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However, the catalytic mutants (C178A and H607A) devoid of DUB activity lost the ability of USP25 WT-mediated IFN inhibition to some degree, indicating that DUB activity is involved in USP25 inhibition of type I IFN induction.To further determine the levels at which USP25 negatively regulates type I IFN signaling, HEK-293T cells were transfected with DNA constructs encoding RIG-I, IPS-1, TRAF2, or TRAF6, together with IFN-β-Luc.

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To examine whether the inhibitory effects of USP25 on SEV-induced type I IFN signaling is due to its deubiquitinase activity, wild-type USP25 (USP25-WT) and its mutants (C178A and H607A) lacking DUB activity were co-transfected with the promoter luciferase reporter plasmid of IFN-β, IRF3, NF-κB and ISRE, and the luciferase activity was detected.

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doi: 10.1371/journal.pone.0080976.g005 USP25 Negatively Regulates Type I IFN Signaling PLOS ONE | www.plosone.org 7B), TRAF2 (Figure 7D), and TRAF6 (The deubiquitinating activity of USP25 is involved in virus-induced type I IFN signaling.
USP25 activates SGCG.
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USP25 activates SGCG. 1 / 1
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However, our studies explain that USP25 negatively modulates RIG-I/MDA5-dependent type I IFN signaling.
USP25 affects GLI1
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USP25 inhibits GLI1.
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USP25 inhibits GLI1. 3 / 3
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In this assay, siRNA mediated depletion of USP21 decreased Gli1 transcriptional activity by ~ 40% (XREF_FIG B).

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USP21 is able to interact with GLI1, thereby suppressing GLI1 dependent transcription.

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Likewise, overexpression of GFP-USP21 inhibited Gli1 transcriptional activity to a similar degree, and this inhibition depended on its catalytic activity (XREF_FIG C).
USP25 activates GLI1.
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USP25 activates GLI1. 3 / 3
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USP21 regulates the Hh signaling pathway and modulates Gli1 transcriptional activity.

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Altogether, these data indicate that USP21 regulates the Hh signaling pathway, and either directly or indirectly modulates Gli1 transcriptional activity.

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USP21 promotes Gli1 localisation at the centrosome.
USP25 decreases the amount of GLI1.
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USP25 decreases the amount of GLI1. 1 / 1
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Depletion of USP21 inhibited SAG dependent accumulation of endogenous Gli1 mRNA levels (XREF_FIG A).
FOXP3 affects USP25
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FOXP3 increases the amount of USP25.
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FOXP3 increases the amount of USP25. 3 / 3
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Interestingly, Foxp3 directly activates expression of USP21, and siRNA knockdown of USP21 downregulates both GATA3 and Foxp3 protein.

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The authors of this study propose that augmentation of USP21 expression by Foxp3 and TCR activation enhances GATA3 levels, which in turn stabilizes Foxp3 function.

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This last report proposed that after TCR stimulation, FOXP3 upregulates USP21 transcription.