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.
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
Transcriptomics
The following table shows the significantly differentially expressed genes after knocking
out USP20 using CRISPR-Cas9.
There were too few differentially expressed genes to run a meaningful GSEA.
Literature Mining
INDRA was used to automatically assemble known mechanisms
related to USP20 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
CLASPIN was also deubiquitinated by bacterially produced GST-USP20, but not by the catalytically inactive mutant GST USP20 (C154S), in the in vitro deubiquitination assays.
These results revealed that USP20 mediated Claspin downregulation did not occur at the gene transcription level, just as the prior study reported that USP20 deubiquitinate and stabilize Claspin.
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].
Here, we report that ubiquitin specific peptidase USP20 deubiquitinates TRAF6 and Tax and suppresses interleukin 1beta (IL-1beta) - and Tax induced NF-kappaB activation.
54 Ubiquitinated beta-arrestin2 promotes NF-kappaB signaling in SMCs, whereas deubiquitinated beta-arrestin2 attenuates NF-kappaB signaling by scaffolding the deubiquitinase USP20, that in turn deubiquitinates TRAF6, which blocks the NF-kappaB signaling cascade.
For example, USP11 negatively regulates TNFα-induced NF-κB activation associated with IκBα and attenuates IκBα degradation [34]; USP20 deubiquitinates TRAF6 and suppresses interleukin 1β (IL-1β)- and Tax-induced NF-κB activation [40]; Katrin et al. showed that USP15 regulates IκBα/NF-κB by deubiquitinylation IκBα[44]; and USP31 inhibits TNFα, CD40, TRAF2, TRAF6 and IKKβ-mediated NF-κB activation [45].
TRAF6 activity can be impeded by deubiquitinating enzymes like ubiquitin specific protease 20 (USP20), which can reverse TRAF6 autoubiquitination, and by association with the multifunctional adaptor protein beta-arrestin2.
With purified proteins and in intact cells, our protein interaction studies showed that TRAF6/USP20 association and subsequent USP20-mediated TRAF6 deubiquitination were β-arrestin2-dependent.
For example, USP11 negatively regulates TNFalpha induced NF-kappaB activation associated with IkappaBalpha and attenuates IkappaBalpha degradation [XREF_BIBR]; USP20 deubiquitinates TRAF6 and suppresses interleukin 1beta (IL-1beta) - and Tax induced NF-kappaB activation [XREF_BIBR]; Katrin et al. showed that USP15 regulates IkappaBalpha and NF-kappaB by deubiquitinylation IkappaBalpha [XREF_BIBR]; and USP31 inhibits TNFalpha, CD40, TRAF2, TRAF6 and IKKbeta mediated NF-kappaB activation [XREF_BIBR].
For example, Junichiro et al. demonstrated that USP21 constitutively deubiquitinates RIP1 in vitro and in vivo [XREF_BIBR]; and a previous study has reported that USP20 deubiquitinates TRAF6 and Tax in vivo [XREF_BIBR].
VDU2 can specifically deubiquitinate and stabilize HIF-1alpha and, therefore, increase expression of HIF-1alpha targeted genes, such as vascular endothelial growth factor (VEGF).
Here, we report that ubiquitin specific peptidase USP20 deubiquitinates TRAF6 and Tax and suppresses interleukin 1beta (IL-1beta) - and Tax induced NF-kappaB activation.
For example, Junichiro et al. demonstrated that USP21 constitutively deubiquitinates RIP1 in vitro and in vivo [XREF_BIBR]; and a previous study has reported that USP20 deubiquitinates TRAF6 and Tax in vivo [XREF_BIBR].
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].
TNF evoked = ~ 2-fold more RIPK1 ubiquitination in SMC-DN-USP20-transgenic than in control SMCs, and RIPK1 was deubiquitinated by purified USP20 in vitro.
In this study, we carried out an unbiased functional loss-of-function screen of the human deubiquitinating enzyme (DUB) family and identified ubiquitin-specific protease 20 (USP20) as a novel ERK3 regulator.| USP20 interacts with and deubiquitinates ERK3 both in vitro and in intact cells.
54 Ubiquitinated beta-arrestin2 promotes NF-kappaB signaling in SMCs, whereas deubiquitinated beta-arrestin2 attenuates NF-kappaB signaling by scaffolding the deubiquitinase USP20, that in turn deubiquitinates TRAF6, which blocks the NF-kappaB signaling cascade.
Similar to Chk1 and Rad17, which enhance recombinational repair of collapsed replication forks, we demonstrate that USP20 depletion impairs DNA double strand break repair by homologous recombination.
CLASPIN was also deubiquitinated by bacterially produced GST-USP20, but not by the catalytically inactive mutant GST USP20 (C154S), in the in vitro deubiquitination assays.
USP20 also deubiquitinates hypoxia inducible factor-1 alpha (HIF-1alpha), promoting HIF-1alpha stability and consequently the expression of its target genes.
Ubiquitinated D2 can be either targeted to proteasomal degradation or reactivated by deubiquitination, a process that is mediated by the deubiquitinases USP20/33 and is important in adaptive thermogenesis.
The ubiquitin specific peptidase USP20 (ubiquitin specific peptidase) deubiquitinates Tax-1 and suppresses IL-1beta- and Tax-1-induced NF-kappaB activation XREF_BIBR.
Given that HERC2 is a component of the replication complex, our data shown in this report suggest that HERC2 and USP20 coordinately modulate CLASPIN stability during S-phase and in response to replication stress.
These results revealed that USP20 mediated Claspin downregulation did not occur at the gene transcription level, just as the prior study reported that USP20 deubiquitinate and stabilize Claspin.
Furthermore, reconstitution of WT USP20 but not catalytically inactive mutant of USP20 (USP20CA) in USP20 depleted cells restored Claspin protein levels.
Furthermore, reconstitution of WT USP20 but not catalytically inactive mutant of USP20 (USP20CA) in USP20 depleted cells restored Claspin protein levels.
Prior studies have demonstrated that USP20 regulate the cell proliferation through the ATK-Claspin-CHK signaling pathway, and downregulation of USP20, could reduce the expression of Claspin through the ubiquitin-proteasome degradation and promote the DNA synthesis, suggesting a defect in the intra-S-phase checkpoint.
Upon replication stress, ATR mediated phosphorylation of USP20 promotes dissociation of HERC2 from USP20, stabilizes USP20 and its association with CLASPIN, thus increasing CALSPIN stability and ensuring CHK1 activation.
To test if USP20 mediated stability of CLASPIN promotes CHK1 activation upon replication stress, we expressed wild-type FLAG-CLASPIN in USP20 depleted cells.
In a candidate screen of ubiquitin specific processing proteases (USPs) for modulating CHK1 activation, we revealed that inhibition of USP20 expression in 293T cells delayed HU induced CHK1 activation, whereas expression of siRNA resistant form of FLAG-USP20res in the endogenous USP20 depleted cells restored HU induced CHK1 activation kinetics.
In line with the clinical outcomes, we found that the overexpression of USP20 could, on one hand, suppress cell proliferation and delay G1-S cell cycle transition, and on the other hand, enhance autophagy in GC cells.
Since overexpression of USP20 inhibits the proliferation of ATL cells, it raises the possibility that induction of USP20 could be used to inhibit HTLV induced oncogenesis.
In HTLV-1-transformed cells, the transcription of USP20 is reduced compared with HTLV-1-negative T cells, and ectopic USP20 expression was found to inhibit the proliferation of an HTLV-1-transformed cell line, MT4.
Depletion of several DUBs caused either resistance or hypersensitivity to HU treatment; among them, USP20 knockdown showed most significant hypersensitivity to HU treatment, while knockdown of USP20 slightly decreased cell proliferation in untreated cells.
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.
Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFkappaB activation by = ~ 50% in response to either TNF or IL-1beta (interleukin-1beta).
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.
USPs are the most abundant DUBs, with approximately 60 proteases in humans. xref USP20 inhibits NF-κB activation via Toll-like receptor (TLR)4, xref IL-1R, and the TNF xref inflammatory pathway in VSMCs.
USP20 is downregulated in HTLV-1 infected cell lines, presumably to promote high levels of NF-kappaB activation, although the exact mechanism is unclear [XREF_BIBR].
Coimmunoprecipitation experiments revealed that USP20 associates with several components of the TNFR1 (TNF receptor-1) signaling pathway, including RIPK1 (receptor interacting protein kinase 1), a critical checkpoint in TNF induced NFkappaB activation and inflammation.
The previous study has shown that Usp20 increases the expression of HIF1A XREF_BIBR, XREF_BIBR, which is reported as a metabolic switch for an early stage of iPSC 20.
In HERC2 depleted cells, USP20 and its substrates Claspin were significantly upregulated and the phosphorylation of CHK1 is more sustained, even at later time points.
Our result found that silencing of USP20 could inhibit autophagy, not singly but in pairs, the overexpression of USP33, which is the homology of USP20, could deubiquitinate RALB at Lys 47, and then regulate assembly of the RALB, EXO84, and beclin1 complex that initiated autophagy.
Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFkappaB activation by = ~ 50% in response to either TNF or IL-1beta (interleukin-1beta).
Coimmunoprecipitation experiments revealed that USP20 associates with several components of the TNFR1 (TNF receptor-1) signaling pathway, including RIPK1 (receptor interacting protein kinase 1), a critical checkpoint in TNF induced NFkappaB activation and inflammation.
These suggest that ATR mediated phosphorylation of USP20 promotes dissociation of USP20 from HERC2, and this dissociation would stabilize CLASPIN and promote CHK1 activation.
] Although deubiquitinating STING alone , USP20 can be recruited by USP18 , thus causing USP20 to remove K48-linked polyubiquitin chains more efficiently , even at a low dose .
Under unperturbed condition, HERC2 ubiquitinates USP20 and promotes ubiquitination mediated proteasomal degradation of USP20, regulating the status of K48 linked polyubiquitination of CLASPIN and ensuring appropriate protein levels of CLASPIN during the S-phase.
Genetic deletion or pharmacological inhibition of USP20 markedly decreases diet induced body weight gain, reduces lipid levels in the serum and liver, improves insulin sensitivity and increases energy expenditure.
As shown in XREF_FIG, the silencing of USP20 expression accelerated the G1-S cell cycle transition which is accompanied by decrease in the percentage of G1 cells (down to 54.05%) and increase in the percentage of cells in the S phase (up to 37.62%), and the tendency was also consistent with MKN45-siUSP20 cells and BGC-823-siUSP20 cells (P < 0.05; XREF_FIG and XREF_FIG).
In line with the clinical outcomes, we found that the overexpression of USP20 could, on one hand, suppress cell proliferation and delay G1-S cell cycle transition, and on the other hand, enhance autophagy in GC cells.
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.
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.
Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFkappaB activation by = ~ 50% in response to either TNF or IL-1beta (interleukin-1beta).
VDU1 and VDU2, a closely related isoform, have been shown to increase half-life of type 2 iodothyronine deiodinase by their deubiquitinating activity [23].
As shown in XREF_FIG, silencing of USP20 significantly promoted cell growth in monolayer culture, and further experimental results revealed that the proliferation ability of MGC-803-USP20 cells were lower than that of MGC-803-NC cells (XREF_FIG).
Because our results suggest that the USP20-p62 axis is responsible for PKCzeta-mediated NF-kappaB activation for cell survival , USP20 depletion may promote cell death .
Notably , suppressing PTEN with its specific inhibitor dramatically abolished the function of USP20 to ameliorate neuroinflammation and neuron death induced by OGD / R. Collectively , our results illustrated that USP20 could effectively mitigate the severity of cerebral ischemic stroke and improve behavior deficits in MCAO-operated mice , and identified the USP20 / PTEN axis as a promising therapeutic target for ischemic stroke treatment .
The closely related DUBs, USP20 and USP33, deubiquitylate activated beta-adrenergic receptors as well as the adaptor and signalling protein beta-arrestin (in the case of USP33), to favour recycling from endosomes, despite largely being associated with the secretory pathway 75, 76, 77.
For example, USP17 and USP20 were found to target RIG-I and TRAF6 respectively, thereby functioning as novel regulators of antiviral innate immune responses [XREF_BIBR, XREF_BIBR].
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.
All these effects mediated by USP20 during cerebral I/R injury were confirmed in the cultured primary microglial cells and cortical neurons stimulated by oxygen-glucose deprivation and reoxygenation (OGD/R).
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.
Genetic deletion or pharmacological inhibition of USP20 markedly decreases diet induced body weight gain, reduces lipid levels in the serum and liver, improves insulin sensitivity and increases energy expenditure.
For example, USP17 and USP20 were found to target RIG-I and TRAF6 respectively, thereby functioning as novel regulators of antiviral innate immune responses [XREF_BIBR, XREF_BIBR].
Furthermore, reconstitution of WT USP20 but not USP20-4 mut rescued the cell viability after UV treatment and restored UV or HU induced intra-S-phase checkpoint.
Lu et al. demonstrated the post-prandial increase in insulin and the glucose concentration stimulates mTORC1 to phosphorylate the deubiquitylase ubiquitin-specific peptidase 20 (USP20) at S132 and S134, which is recruited to the HMGCR complex and antagonizes its degradation (Lu et al., 2020).
147 In addition , USP20 attenuated atherosclerosis in an LDLr - / - mouse model by inhibiting NF-kappaB activation in vivo.148 Therefore , increased expression / activity of USP20 may offer protection against inflammatory pathways in patients with ISR .
Nevertheless, in USP20 knockdown cells, reconstitution of Claspin was able to rescue phospho-CHK1 levels and significantly suppress tumorigenesis, even if Rad17 levels was decreased, suggesting that Claspin is an important target of USP20 in mediating checkpoint activation and tumor suppression.
Notably, suppressing PTEN with its specific inhibitor dramatically abolished the function of USP20 to ameliorate neuroinflammation and neuron death induced by OGD/R.
Concomitant inhibition of the deubiquitinase USP20 (Ubiquitin-specific-processing protease 20) by PKA favors ubiquitylation and degradation of the receptor by NEDD4 ( xref ; xref ).
Our experiments showed that hVDU2 protein was extremely stable in the group without co-transfection of pVHL during cycloheximide treatment, and MG132 did not significantly increase the level of VDU2 p[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Mechanistically, ERK3 expression level in cancer cells was shown to be upregulated by BRAF (through increasing ERK3 mRNA) 11, BMI1 (by suppressing let-7i which targets ERK3 mRNA) 12 and USP20 (by deubiquitinating and stabilizing ERK3 protein) 13.
Nevertheless, in USP20 knockdown cells, reconstitution of Claspin was able to rescue phospho-CHK1 levels and significantly suppress tumorigenesis, even if Rad17 levels was decreased, suggesting that Claspin is an important target of USP20 in mediating checkpoint activation and tumor suppression.
We found that overexpression of FLAG-CLASPIN corrected the delay of USP20 depletion-induced CHK1 activation in response to HU treatment (Figure xref ).
VDU1, but not VDU2, is markedly increased in brown adipocytes by norepinephrine or cold exposure, further amplifying the increase in D2 activity that results from catecholamine stimulated de novo synthesis.