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 USP12 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 USP12 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
USP12 in the presence of its cofactors Uaf-1 and WDR20 deubiquitinates AR to enhance AR protein stability and transcriptional activity [71] USP12 in complex with Uaf-1 and WDR20 also abrogates phospho[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The AR is known to be regulated by a number of post-translational modifications and we have recently identified the deubiquitinating enzyme Usp12 as a positive regulator of AR.
In this report we demonstrate that Usp12, in complex with Uaf-1 and WDR20, deubiquitinates the AR to enhance receptor stability and transcriptional activity.
Both USP12 and USP46 deubiquitinate histone H2A and H2B, affecting Xenopus development (Joo et al., 2011), and, unlike USP1, these smaller USPs can simultaneously bind WDR20 in addition to WDR48 (Joo [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Joo et al. demonstrated that USP46 and USP12 containing fractions from HeLa cells can deubiquitinate histones H2A and H2B to control cell fate and gastrulation during Xenopus development [XREF_BIBR].
we demonstrate that Usp12, in complex with Uaf-1 and WDR20, directly deubiquitinates and stabilises the Akt phosphatases PHLPP and PHLPPL resulting in decreased levels of active pAkt.
Collectively, our study identified that USP12 is responsible for deubiquitinating and stabilizing MDK and leads to metastasis by promoting angiogenesis.
Joo et al. demonstrated that USP46 and USP12 containing fractions from HeLa cells can deubiquitinate histones H2A and H2B to control cell fate and gastrulation during Xenopus development [XREF_BIBR].
Knock-down of USP46, but not USP12, results in increased levels of ubiquitinated GluA1, decreased surface and total levels of GluA1, and reduced mEPSC amplitudes, consistent with a role for USP46 in deubiquitinating mammalian AMPARs (Huo et al., 2015).
Similarly, the ubiquitin specific peptidase 12 (USP12) can deubiquitinate LAT at the proximal TCR (Figure 2), to protect it from ubiquitin-dependent lysosomal degradation [146].
Similarly, the ubiquitin specific peptidase 12 (USP12) can deubiquitinate LAT at the proximal TCR (Figure 2), to protect it from ubiquitin-dependent lysosomal degradation [146].
Wild type UAF1 was unable to activate USP12 E190K to similar levels as compared to USP12 WT ( xref d, e) and similarly, the UAF1 3X mutant did no longer activate.
To delineate the mechanism by which UAF1 activates USP12, we next determined the crystal structure of USP12 in complex with full-length human UAF1 in F222 space group at 3.2 A (XREF_SUPPLEMENTARY).
Activation of USP12 by UAF1 requires structural fine tuning at the junction of the Ub globular domain and it tail through BL1, whereas WDR20 acts independently of this site and directly affects the ca[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Activation of USP12 by UAF1 requires structural fine-tuning at the junction of the Ub globular domain and it tail through BL1, whereas WDR20 acts independently of this site and directly affects the ca[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
For example, USP1, USP12, and USP46 are activated by the WD40-repeat containing UAF1, and USP7 is activated by GMPS (Cohn et al., 2007, 2009; Faesen et al., 2011; van der Knaap et al., 2005).
Wild type UAF1 was unable to activate USP12 E190K to similar levels as compared to USP12 WT (XREF_FIG d, e) and similarly, the UAF1 3X mutant did no longer activate.
The E1 protein from anogenital HPV types interacts with the UAF1 associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome.
To investigate the mechanism by which WDR20 activates USP12 , we next determined the crystal structure of a ternary complex consisting of USP12 , UAF1 lacking its SLD , and WDR20 at a 3 .
These results strongly suggest that WDR20 can activate USP12 by allosterically optimizing its catalytic cleft, whereas the effect of UAF1 binding can not be directly transmitted to the catalytic cysteine through USP12.
Although mammalian USP46 and USP12 DUB activity can be stimulated by two proteins UAF-1 and WDR20, the C. elegans homologs of these genes have not yet been identified.
In agreement with our enzyme kinetics data, our binding studies revealed negligible changes in the substrate binding affinity of USP12 induced by UAF1 or WDR20 (XREF_FIG and XREF_SUPPLEMENTARY).
These results suggest that even though fine-tuning the Ub globular domain-tail junction is not critical for WDR20 to activate USP12 , interaction between BL1 and the Ub globular domain is still important for the full WDR20-stimulated USP12 activity .
In agreement with our enzyme kinetics data, our binding studies revealed negligible changes in the substrate binding affinity of USP12 induced by UAF1 or WDR20.
In agreement with our enzyme kinetics data, our binding studies revealed negligible changes in the substrate binding affinity of USP12 induced by UAF1 or WDR20 (Figures 6A and S6) .
Overall, our two UAF1-USP12 structures not only reveal a series of structural changes of the enzyme that are connected to the binding site of UAF1 but also highlight the dynamic nature of three key lo[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
In USP12, this loop is important for activation, as mutation of a stretch of glycine residues within the loop leads to loss in activation of USP12 by either UAF1 or WDR20.
Cell lines used in this study express low levels of endogenous Usp12 and Uaf-1, consequently even without the addition of Uaf-1 overexpression of WDR20 alone was enough to significantly enhance the AR activity.
Our previous observations for the LNCaP cell line that express AR FL but not AR Vs have demonstrated that USP12 silencing causes a decrease in transcript expression of all the AR target genes [XREF_BIBR] supporting our data that AR Vs are not targeted by USP12 in the AR V expressing CWR22Rv1 cell line.
This further confirms that each complex member alone has a limited effect on AR activity but in combination the Usp12 complex significantly increases AR transcriptional activity implying that targeting any of the binding partners should offer the same efficacy as targeting Usp12 alone.
We have recently reported that Usp12, in combination with Uaf-1, can increase the transcriptional activity of AR [XREF_BIBR], and hence we wanted to analyse the importance of Akt phosphorylation sites in this process.
Interestingly, Usp12 and WDR20 alone significantly increased AR activity but this effect was greatest when Usp12, Uaf-1 and WDR20 were overexpressed simultaneously.
Also, USP12 co-localizes with AR in the cytoplasm and promotes AR transcriptional activity by confronting the ubiquitin dependent degradation of AR [XREF_BIBR].
Also, USP12 co-localizes with AR in the cytoplasm and promotes AR transcriptional activity by confronting the ubiquitin dependent degradation of AR [XREF_BIBR].
Our previous observations for the LNCaP cell line that express AR FL but not AR Vs have demonstrated that USP12 silencing causes a decrease in transcript expression of all the AR target genes [XREF_BIBR] supporting our data that AR Vs are not targeted by USP12 in the AR V expressing CWR22Rv1 cell line.
Usp12 stimulation of autophagy, in the context of mHTT, could lead to more efficient cargo loading in autophagosomes, and thereby reduce mHTT associated toxicity.
Both alpha-synuclein and TDP-43 are substrates for autophagic degradation XREF_BIBR, XREF_BIBR, XREF_BIBR, and so, we might predict that stimulation of autophagy by Usp12 overexpression would also be neuroprotective in the related PD and ALS neuron models.
Publisher Correction : Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington 's disease.
Publisher Correction : Deubiquitinase Usp12 functions noncatalytically to induce autophagy and confer neuroprotection in models of Huntington 's disease.
As a result, depleting Usp12 decreased PC cellular proliferation and increased cellular apoptosis suggesting it may be a potential target for CRPC therapy [XREF_BIBR].
As shown in XREF_FIG B, knockdown of WDR48 and USP12 by shRNA accelerated the rate of cell proliferation compared with control shRNA transfected cells.
We further established that Usp12 depletion reduced PC cell proliferation and induced apoptosis, furthermore Usp12 levels are increased in PC tissue [XREF_BIBR].
As a result, depleting Usp12 decreased PC cellular proliferation and increased cellular apoptosis suggesting it may be a potential target for CRPC therapy [XREF_BIBR].
In vivo experiments showed that USP12-knockdown could suppress tumor growth in mice , and immuno-blotting revealed that USP12 could induce G2 / M arrest through the cyclin dependent kinase 1 / cyclinB1 axis , and trigger apoptosis via the p38 / mitogen-activated protein kinase pathway .
Invivo experiments showed that USP12-knockdown could suppress tumor growth in mice, and immuno blotting revealed that USP12 could induce G2/M arrest through the cyclin dependent kinase1 and cyclinB1 axis, and trigger apoptosis via the p38 and mitogen activated protein kinase pathway.
We further established that Usp12 depletion reduced PC cell proliferation and induced apoptosis, furthermore Usp12 levels are increased in PC tissue [XREF_BIBR].
The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7 and TGF-beta signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1 and TGF-betaRI, and daf4 and R-SMAD, but not of daf-8 and R-SMAD.
Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7 and TGF-beta signaling, suggesting that this mode of regulation of TGF-beta signaling is conserved across animal species.
Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7 and TGF-beta signaling, suggesting that this mode of regulation of TGF-beta signaling is conserved across animal species.
The dauer larva-constitutive C. elegans phenotype caused by defective DAF-7 and TGF-beta signaling was enhanced and suppressed, respectively, by ubh-1 deletion and overexpression in the loss-of-function genetic backgrounds of daf7, daf-1 and TGF-betaRI, and daf4 and R-SMAD, but not of daf-8 and R-SMAD.
Our data demonstrates that Usp12 increases the levels of PHLPP and PHLPPL that in turn decreases the active pAkt pool and inhibits AR S213 phosphorylation by Akt.
Importantly, overexpression of Usp12 deubiquitinated both PHLPP and PHLPPL and this elevated the steady-state levels of the enzymes, while overexpression of an enzymatically inactive Usp12 C48A mutant failed to elevate PHLPP and PHLPPL levels suggesting the importance of Usp12 enzymatic activity for phosphatase regulation.
Because PHLPP1 is a potent negative regulator of the PI3-kinase and Akt pathway, we hypothesized that WDR48 and USP12 might suppress Akt signaling by stabilizing PHLPP1.
Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7 and TGF-beta signaling, suggesting that this mode of regulation of TGF-beta signaling is conserved across animal species.
Here, we show that the deubiquitinating enzyme UBH-1 in Caenorhabditis elegans and its human homolog, ubiquitin C-terminal hydrolase-L1 (UCH-L1), stimulate DAF-7 and TGF-beta signaling, suggesting that this mode of regulation of TGF-beta signaling is conserved across animal species.
As knockdown of WDR48 and USP12 increased Akt activation, we next tested whether WDR48 and USP12 could potentiate the suppression of Akt signaling by PHLPP1.
Because PHLPP1 is a potent negative regulator of the PI3-kinase and Akt pathway, we hypothesized that WDR48 and USP12 might suppress Akt signaling by stabilizing PHLPP1.
Finally, our data show that USP12 is partially dependent on the stabilization of p300 to activate METTL3 expression and promote myocardial hypertrophy.
Our data demonstrates that Usp12 increases the levels of PHLPP and PHLPPL that in turn decreases the active pAkt pool and inhibits AR S213 phosphorylation by Akt.
Importantly, overexpression of Usp12 deubiquitinated both PHLPP and PHLPPL and this elevated the steady-state levels of the enzymes, while overexpression of an enzymatically inactive Usp12 C48A mutant failed to elevate PHLPP and PHLPPL levels suggesting the importance of Usp12 enzymatic activity for phosphatase regulation.
The Ubiquitin specific Protease 12 (USP12) is a negative regulator of Notch signaling as USP12 directly targets Notch and directs it to lysosomal degradation.
Usp12 and Usp12-C48S overexpression significantly decreased the mean half-life of LC3, but did not affect the half-life of the control protein Dendra2.
Conversely, Usp12 knockdown increased the mean half-life of LC3, while also significantly right shifting the distribution of LC3 half-lives in individual neurons.
Since a non catalytic mutant of Usp12, Usp12-C48S, stimulated LC3 clearance as efficiently as Usp12, we additionally conclude that this function of Usp12 is independent of its deubiquitinating activity.
Identification of Usp12 as a modifier of HD suggested that Usp12 could rescue neurodegeneration in related disorders, such as amyotrophic lateral sclerosis (ALS) and Parkinson 's disease (PD) that are characterized by proteotoxic stress.
In C. elegans, USP46 and USP12 both bind to USP1 associated factor 1 (UAF1 and WDR48) and the binding dramatically enhances the activity of USP12 and USP46.
Most of the deubiquitinases, including A20 and CYLD, can inhibit NF-kappaB activation [XREF_BIBR], while USP12 positively regulates the LPS signal [XREF_BIBR].
Knockdown of USP12 decreased the lung metastasis ability of 4T1 cells, while USP12 overexpression increased the lung metastasis ability of these cells in vivo.
Collectively , our study identified that USP12 is responsible for deubiquitinating and stabilizing MDK and leads to metastasis by promoting angiogenesis .
Knock-down of USP46, but not USP12, results in increased levels of ubiquitinated GluA1, decreased surface and total levels of GluA1, and reduced mEPSC amplitudes, consistent with a role for USP46 in deubiquitinating mammalian AMPARs.
Knock-down of USP46, but not USP12, results in increased levels of ubiquitinated GluA1, decreased surface and total levels of GluA1, and reduced mEPSC amplitudes, consistent with a role for USP46 in deubiquitinating mammalian AMPARs (Huo et al., 2015).
USP12 in the presence of its cofactors Uaf-1 and WDR20 deubiquitinates AR to enhance AR protein stability and transcriptional activity [71] USP12 in complex with Uaf-1 and WDR20 also abrogates phospho[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
For example, GMP synthetase (GMPS) interacts and activates USP7 (Faesen et al., 2011; Sarkari et al., 2009; van der Knaap et al., 2005), whereas the WD40 repeat containing UAF1 (WDR48) activates USP1,[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Highlights d Free USP12 deubiquitinase is inactive and has several flexible structural elements d UAF1 and WDR20 can both activate USP12 without increasing its substrate affinity d UAF1 and WDR20 bind USP12 at two distinct sites away from its catalytic center d Two distinct allosteric mechanisms underlie USP12 activation by UAF1 and WDR20 Ubiquitin-specific proteases ( USPs ) constitute the largest family of deubiquitinating enzymes , whose catalytic competency is often modulated by their binding partners through unknown mechanisms .
In C. elegans, USP46 and USP12 both bind to USP1 associated factor 1 (UAF1 and WDR48) and the binding dramatically enhances the activity of USP12 and USP46.
Thus, USP12 and OTUD7B counterbalance inhibitory E3 ligases to stabilize TCR signaling.Aside from acting at the proximal signalosome, E3 ligases also inhibit T-cell activation by acting further downstream, controlling the key transduction events that lead to the nuclear translocation of the T-cell activating transcription factors.
The largely constant substrate affinity of USP12 in different forms negates a role of the BLs in controlling the accessibility of the Ub-binding surface of the enzyme.
The partial overlap of the binding interfaces and the high affinity of WDR20 suggests that WDR20 binding to USP12 and UAF1 can either prevent binding of the second UAF1 molecule or it can actively compete out UAF1 if it is bound to the USP12 and UAF1 complex.
Consistent with an important role in binding USP12, mutations of W306 and F262 effectively abolish the ability of WDR20 to interact with and activate USP12 ( Figures 4C and S4D ).
Our previous observations for the LNCaP cell line that express AR FL but not AR Vs have demonstrated that USP12 silencing causes a decrease in transcript expression of all the AR target genes [XREF_BIBR] supporting our data that AR Vs are not targeted by USP12 in the AR V expressing CWR22Rv1 cell line.
Also, USP12 co-localizes with AR in the cytoplasm and promotes AR transcriptional activity by confronting the ubiquitin dependent degradation of AR [XREF_BIBR].
In C. elegans, USP46 and USP12 both bind to USP1 associated factor 1 (UAF1 and WDR48) and the binding dramatically enhances the activity of USP12 and USP46.
In contrast, USP12 has been found to stabilize the TCR complex and promote TCR signaling through deubiquitylating TCR adaptor proteins LAT and Trat1 in primary mouse T lymphocytes.
Thus, USP12 and OTUD7B counterbalance inhibitory E3 ligases to stabilize TCR signaling.Aside from acting at the proximal signalosome, E3 ligases also inhibit T-cell activation by acting further downstream, controlling the key transduction events that lead to the nuclear translocation of the T-cell activating transcription factors.
Other studies have shown that USP12 suppresses the PH domain and leucine rich repeat protein phosphatases, which restrains the activation of macrophages through the dephosphorylation of transcription factor STAT1 [XREF_BIBR].
In vivo experiments showed that USP12-knockdown could suppress tumor growth in mice , and immuno-blotting revealed that USP12 could induce G2 / M arrest through the cyclin dependent kinase 1 / cyclinB1 axis , and trigger apoptosis via the p38 / mitogen-activated protein kinase pathway .
Additionally, the study also found out that the UAF1/USP12 and UAF1/USP46 complexes increase p65 expression, which promotes NF-κB activation and increases NLRP3 and IL-1β expression levels.
In this study, we showed that knockdown of USP12 effectively induced cell cycle arrest in HeLa cells and decreased BMI-1, c-Myc and cyclin D2 transcription levels.
Invivo experiments showed that USP12-knockdown could suppress tumor growth in mice, and immuno blotting revealed that USP12 could induce G2/M arrest through the cyclin dependent kinase1 and cyclinB1 axis, and trigger apoptosis via the p38 and mitogen activated protein kinase pathway.
Thus, in this study we probed the involvement of USP12 in macrophage mediated inflammatory responses using bacterial endotoxin, LPS, as the model system.
For example, USP26 physically interacts with AR and influences AR ubiquitination and transcriptional activation; 17 USP12 stabilizes AR, enhances its cellular function, and thereby triggers the gene expression of PSA.
We observed that siRNA mediated ablation of USP12 expression in mouse macrophages suppressed the induction of LPS induced iNOS and IL-6 expression but failed to alter IFN-beta synthesis, oxidative stress and phagocytic ability of macrophages.
Additionally, the study also found out that the UAF1/USP12 and UAF1/USP46 complexes increase p65 expression, which promotes NF-κB activation and increases NLRP3 and IL-1β expression levels.
In this study, we showed that knockdown of USP12 effectively induced cell cycle arrest in HeLa cells and decreased BMI-1, c-Myc and cyclin D2 transcription levels.
In this study, we showed that knockdown of USP12 effectively induced cell cycle arrest in HeLa cells and decreased BMI-1, c-Myc and cyclin D2 transcription levels.
XREF_BIBR, XREF_BIBR In addition, other E3 ubiquitin ligases are probably involved in the AR regulation process, such as CHIP XREF_BIBR and SKP2 XREF_BIBR which also promote AR degradation, and Siah2, XREF_BIBR RNF6, XREF_BIBR and USP12 XREF_BIBR, XREF_BIBR which promote deubiquitination and AR activation.
XREF_BIBR, XREF_BIBR In addition, other E3 ubiquitin ligases are probably involved in the AR regulation process, such as CHIP XREF_BIBR and SKP2 XREF_BIBR which also promote AR degradation, and Siah2, XREF_BIBR RNF6, XREF_BIBR and USP12 XREF_BIBR, XREF_BIBR which promote deubiquitination and AR activation.
Highlights d Free USP12 deubiquitinase is inactive and has several flexible structural elements d UAF1 and WDR20 can both activate USP12 without increasing its substrate affinity d UAF1 and WDR20 bind USP12 at two distinct sites away from its catalytic center d Two distinct allosteric mechanisms underlie USP12 activation by UAF1 and WDR20 Ubiquitin-specific proteases ( USPs ) constitute the largest family of deubiquitinating enzymes , whose catalytic competency is often modulated by their binding partners through unknown mechanisms .
Because we did not find peptides corresponding to other EBNA proteins in these complexes, each EBNA3 protein appears to target RBPJ and the USP46 and USP12 DUB complexes independently.