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.
There were too few differentially expressed genes to run a meaningful GSEA.
Literature Mining
INDRA was used to automatically assemble known mechanisms
related to BRCC3 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
Altogether, these results demonstrate that BRCC3 mediates the deubiquitination of the LRR domain of NLRP3, an important regulatory mechanism in the activation of NLRP3 inflammasome by its cognate stim[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Viral RNA (vRNA) molecule-induced NLRP3 inflammasome activation also comes about by post-translational priming, but involving the RIP1/caspase 8/RIP3 signaling pathway (29, 45), which may ultimately result in the deubiquitination of NLRP3 protein by BRCC3 (46, 47).
The K-63-specific deubiquitinating enzyme BRCC3 mediates the deubiquitylation of NLRP3, which has recently been shown to occur in response to pattern recognition receptor stimulation [XREF_BIBR].
Moreover, BRCC3 [18], STING [19] and ABRO1 [20] can be activated by NLRP3 agonists, such as ATP, to promote the deubiquitination and sensitivity of NLRP3.
By screening a deubiquitinating enzyme (DUB) library, they recognized that BRCC3 (BRCA1, BRCA2, and containing complex subunit 3) deubiquitinates the LRR domain of NLRP3, which then proceeds NLRP3 activation 59.
NLRP3 deubiquitination is mediated by BRCC3, a member of JAMM domain containing Zn 2+ metalloprotease deubiquitinating enzyme family, therefore providing a novel therapeutic target for IL-1beta-associated inflammatory diseases.
In the BRCC36 isopeptidase complex, BRCC36 promotes interferon dependent responses by deubiquitinating and stabilizing the type I interferon receptor XREF_BIBR.
Dr. Greenberg group from University of Pennsylvania showed that BRCC36 containing deubiquitinating complex BRISC which is also a sister protein complex of nuclear RAP80-BRCA1 complex, deubiquitinates type I interferon receptor (IFNAR1), resulting in its delayed lysosomal dependent degradation.
The Abraxas complex (ARISC) functions in DNA repair and breast cancer suppression, while the BRCC36 isopeptidase complex (BRISC) promotes interferon dependent responses by deubiquitinating and stabilizing the type I interferon receptor, IFNAR1.
In addition, consistent with its role as a deubiquitination enzyme for NLRP3, BRCC3 expression significantly reduced the extent of NLRP3 covalent ubiquitination in 293T cells.
BRCA1/2-containing complex36 (BRCC36) is one of the DUBs which localizes at the site of DSBs as a part of the RAP80 complex and deubiquitinates RNF8-dependent γHA2X ubiquitination
importantly, BRISC promotes the assembly of functional bipolar spindle by deubiquitinating the essential spindle assembly factor nuclear mitotic apparatus (NuMA).
To identify the DUB associated with NALP7, we over-expressed plasmids expressing several candidate DUBs including BRCC3, which deubiquitinates NALP3, COPS5, STAMBPL1, USP7 and the endosome associated DUBs, STAMBP and USP8.
Dr. Greenberg group from University of Pennsylvania showed that BRCC36 containing deubiquitinating complex BRISC which is also a sister protein complex of nuclear RAP80-BRCA1 complex, deubiquitinates type I interferon receptor (IFNAR1), resulting in its delayed lysosomal dependent degradation.
Recently, Py and colleagues demonstrated that BRCC3, a JAMM domain containing zinc metalloprotease DUB, promotes NLRP3 inflammasome activation by deubiquitinating the mixed K64 and K48 ubiquitin chains on both the NACHT and LRR domains of NLRP3 [XREF_BIBR].
There are many examples of HDACs being recruited to sites of DNA repair (as discussed above), and the BRCA-1A complex actually contains a DUB, Brcc36, that can deubiquitinate histones at least in vitro.
Dr. Greenberg group from University of Pennsylvania showed that BRCC36 containing deubiquitinating complex BRISC which is also a sister protein complex of nuclear RAP80-BRCA1 complex, deubiquitinates type I interferon receptor (IFNAR1), resulting in its delayed lysosomal dependent degradation.
Recently, Py and colleagues demonstrated that BRCC3, a JAMM domain containing zinc metalloprotease DUB, promotes NLRP3 inflammasome activation by deubiquitinating the mixed K64 and K48 ubiquitin chains on both the NACHT and LRR domains of NLRP3 [XREF_BIBR].
To identify the DUB associated with NALP7, we over-expressed plasmids expressing several candidate DUBs including BRCC3, which deubiquitinates NALP3, COPS5, STAMBPL1, USP7 and the endosome associated DUBs, STAMBP and USP8.
To identify the DUB associated with NALP7, we over-expressed plasmids expressing several candidate DUBs including BRCC3, which deubiquitinates NALP3, COPS5, STAMBPL1, USP7 and the endosome associated DUBs, STAMBP and USP8.
We show here that a LNK-associated lysine-63 (K63)-deubiquitinating enzyme complex, Brcc36 isopeptidase complex (BRISC), attenuates HSC expansion through control of JAK2 signaling.
Recently, Py and colleagues demonstrated that BRCC3, a JAMM domain containing zinc metalloprotease DUB, promotes NLRP3 inflammasome activation by deubiquitinating the mixed K64 and K48 ubiquitin chains on both the NACHT and LRR domains of NLRP3 [XREF_BIBR].
Recently, BRCC3 in the cytosol has been reported to promote the inflammasome activation by deubiquitinating NLRP3 in LPS primed macrophages [XREF_BIBR].
Besides phosphorylation, the deubiquitinating enzyme BRCA1/BRCA2-containing complex subunit 3 (BRCC3) promotes an inflammasome activation by deubiquitinating NLRP3 at its LRR domain 153 .
The study has demonstrated that BRCC3 depletion prevents the formation of BRCA1 nuclear foci, and subsequently impairs the DNA repair pathway in response to DNA damage by ionizing radiation in breast cancer cells, suggesting that BRCC3 is referred as a potential therapeutic target for breast cancer [XREF_BIBR].
Immunofluorescent staining of BRCA1 and gamma-H2AX indicates that BRCC36 depletion prevents the formation of BRCA1 nuclear foci in response to DNA damage in breast cancer cells.
The so called BRCA1-A complex comprises RAP80, Abraxas and CCDC98, the deubiquitinase BRCC36, BRCC45 and MERIT40, which are thought to target BRCA1 to IR induced foci through the interaction of RAP80 with polyubiquitin chains such as is thought to occur at histone gamma-H2AX [XREF_BIBR, XREF_BIBR] (see XREF_FIG).
But beside the canonical role of deubiquitinating enzymes in antagonizing ubiquitin ligase activity, BRCC36 also enhances BRCA1 and BARD1 E3 ubiquitin ligase activity.
Previous structural studies have clearly shown how the BRISC subunit Abro1 binds to and allosterically activates the DUB activity of BRCC36 and how this heterodimer further self-associates to form a " superdimeric " assembly.
Interestingly, although CCDC98 functions as an adaptor of BRCC36 and regulates BRCC36 activity in the nucleus, KIAA0157 mainly localizes in cytosol and activates BRCC36 in the cytoplasm.
We pursued crystallization of BRCC36-KIAA0157 subcomplexes from different species (H. sapiens, G. gallus, X. tropicalis, D. rerio, C. floridanus and A. thaliana) to determine the structural basis for (1) how KIAA0157 supports the catalytic function of BRCC36, and (2) how super dimerization of the minimally active BRCC36 and KIAA0157 heterodimer is mediated.
This likely reflects the fact that the mechanism by which KIAA0157 and Abraxas support BRCC36 will differ from how CSN6 and RPN8 support and regulate the function of CSN5 and RPN11.
The authors further suggested that the deubiquitylation of NLRP3 was critical for the inflammasome activation based on the facts that inhibiting BRCC3 could abolish NLRP3 inflammasome activation under a diverse range of classic " activation signals, " including K + efflux, ROS overproduction, and lysosomal destabilization.
Deubiquitination of NLRP3 by the BRCA1 / BRCA2 containing complex subunit 3 (BRCC3) promoted NLRP3 inflammasome activity by preventing its degradation 39.
BRCC3 knockdown leads to an increase in NLRP3 ubiquitination, and the DUB activity of BRCC3 is required for caspase-1 activation and IL-1beta processing, but not for LPS induced transcription of pro-IL-1beta or of NLRP3 itself XREF_BIBR.
Functionally, we show that lentivirus mediated overexpression of WWP2 in murine macrophages inhibits NLRP3 inflammasome activation by decreasing BRCC3 protein level.
In contrast, the knockdown of BRCC3 (BRCC3-KD) through the application of lentivirus mediated shRNA delivery system into OPCs suppressed OL differentiation by decreasing MBP expression and PLP production.
BRCC3 siRNA promoted differentiation of osteoblasts and up-regulated beta-catenin After transfected with BRCC3 siRNA and cultured for 21 days, the cells were stained with ALP and ARS (XREF_FIG).
Moreover, depletion of BRCC3 promoted osteogenic differentiation of osteoblast through up-regulated beta-catenin expression, whereas opposite effects were observed after BRCC3 overexpression.
Results showed that XAV939 inhibited the ALP activation and formation of calcified nodules induced by BRCC3 siRNA, suggesting that beta-catenin signaling mediated the BRCC3 siRNA induced osteogenic differentiation (XREF_FIG).
Inhibition of beta-catenin reversed BRCC3 siRNA induced osteogenic differentiation To test the role of beta-catenin in BRCC3 siRNA actions, cells were treated with XAV939 to inhibit the activation of beta-catenin signaling.
Knockdown of BRCC3 increased the cell survival fraction, attenuated DNA damage repair and resulted in G2/M cell cycle arrest in radioresistant NPC cells.
Our data showed that BRCC3 gene knockdown reduced the cell viability of TMZ treated U251 and A172 cells when compared to the relative TMZ treated mock cells.
The inhibition of BRCC3 gene expression significantly reduces the cell viability and clonogenicity of U251 and A172 cells by TMZ treatment, indicating that BRCC3 can be treated as the target in order to enhance the sensitivity of glioma cells to TMZ.
Furthermore, BRCC3 interference inhibited the cell viability, invasion and migration abilities of HeLa and SiHa cells via regulation of EMT progression and expression levels of Snai family members.
Depletion of BRCC3 promoted the activation of alkaline phosphatase and formation of calcified nodules in osteoblasts isolated from osteoporosis patients and up-regulated beta-catenin expression.
Consistently, depletion of BRCC3 increased the level of beta-catenin and promoted osteogenic differentiation, and inhibitor XAV939 inhibited the BRCC3 induced osteogenic differentiation.
Interestingly, we revealed that BRCC36 knockdown decreased beta-catenin expression in CRC cells, suggesting that binding between SHMT2 and BRCC36 may direct BRCC36 activity towards K63-Ub chains conjugated to beta-catenin in CRC cells.
BRCC3 siRNA promoted differentiation of osteoblasts and up-regulated beta-catenin After transfected with BRCC3 siRNA and cultured for 21 days, the cells were stained with ALP and ARS (XREF_FIG).
In 2016, Sun et al[62] reported in their in vitro study that overexpression of B7-H3 upregulates BRCA1/BRCA2-containing complex subunit 3 (BRCC3) expression and antagonizes the anticancer activity of 5-fluorouracil (5-FU).
Altogether, results suggest that BRCC3 may play a role in B7-H3-induced 5-Fu resistance, such that B7-H3 upregulates BRCC3 expression, enhancing DNA repair in colorectal cancer cells.
In addition, a few other mechanisms may also underlie B7H3 mediated chemoresistance : (1) B7H3 induces oxaliplatin resistance by increasing the expression of XRCC1 via PI3K and AKT pathway; (2) B7H3 also enhances cell resistance to chemotherapy by increasing the expression of BRCC3, which antagonizes DNA damage caused by 5-FU; (3) or via the activation of the PI3K and AKT pathway.
Our results indicate that high BRCC3 expression activates NF-kappaB signaling by targeting TRAF2 for activation, which in turn facilitates tumorigenesis in bladder cancer.
Since transduction of wild-type BRCC3, but not of the inactive mutant BRCC3 H122Q, rescued caspase-1 cleavage and IL-1beta secretion inhibited by BRCC3 KD, we conclude that BRCC3 regulates NLRP3 throu[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Particularly, Benedicte et al. found that, knockdown of BRCC3 (a component of the BRISC complex) completely inhibited caspase-1 cleavage and IL-1beta secretion.
Since transduction of wild-type BRCC3, but not of the inactive mutant BRCC3 H122Q, rescued caspase-1 cleavage and IL-1beta secretion inhibited by BRCC3 KD, we conclude that BRCC3 regulates NLRP3 throu[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Altogether, results suggest that BRCC3 may play a role in B7-H3-induced 5-Fu resistance, such that B7-H3 upregulates BRCC3 expression, enhancing DNA repair in colorectal cancer cells.
Thus, the inhibition of BRCC3 expression may impair DNA repair mechanism in U251 and A172 cells, which can promotes sensitization of the two glioma cell lines to TMZ.
In addition, the deficiency of BRCC3 gene expression reduced the levels of DNA repair associated genes (i.e. BRCA1, BRCA2, RAD51 and FANCD2) in TMZ treated U251 and A172 cells, when compared to those detected in TMZ treated mock cells.
In addition, a few other mechanisms may also underlie B7H3 mediated chemoresistance : (1) B7H3 induces oxaliplatin resistance by increasing the expression of XRCC1 via PI3K and AKT pathway; (2) B7H3 also enhances cell resistance to chemotherapy by increasing the expression of BRCC3, which antagonizes DNA damage caused by 5-FU; (3) or via the activation of the PI3K and AKT pathway.
Knockdown of BRCC3 increased the cell survival fraction, attenuated DNA damage repair and resulted in G2/M cell cycle arrest in radioresistant NPC cells.
Given the fact that BRCC3 (BRCC36) is essential for the protection of breast cancer cells from ionizing radiation induced DNA damage [XREF_BIBR], a high level of BRCC3 could protect U251 and A172 cells from TMZ induced cytotoxicity.
Particularly, Benedicte et al. found that, knockdown of BRCC3 (a component of the BRISC complex) completely inhibited caspase-1 cleavage and IL-1beta secretion.
Therefore, we conclude that BRCC3 might be involved in regulating deubiquitination of NLRP3.Consistent with an activator of NLRP3 activity, knockdown of BRCC3 inhibited mature IL-1beta and activated c[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Since transduction of wild-type BRCC3, but not of the inactive mutant BRCC3 H122Q, rescued caspase-1 cleavage and IL-1beta secretion inhibited by BRCC3 KD, we conclude that BRCC3 regulates NLRP3 throu[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Since transduction of wild-type BRCC3, but not of the inactive mutant BRCC3 H122Q, rescued caspase-1 cleavage and IL-1beta secretion inhibited by BRCC3 KD, we conclude that BRCC3 regulates NLRP3 throu[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Our results indicate that high BRCC3 expression activates NF-kappaB signaling by targeting TRAF2 for activation, which in turn facilitates tumorigenesis in bladder cancer.
Our findings showed BRCC3 plays a crucial role in facilitating the development and progression of bladder cancer.To reveal the way how BRCC3 promotes carcinogenesis, we analyzed BRCC3-deficient bladder cancer cell lines by RNA-Seq, and the data showed that the NF-κB inflammatory pathway was notably downregulated when BRCC3 was knockout.
Further analysis showed that knockdown of SHMT1,2 or BRCC36 reduced Tat protein stability without affecting mRNA levels (XREF_SUPPLEMENTARY, XREF_SUPPLEMENTARY).
Taken together, these data suggested a model through which SHMT2 and BRCC36 act to reverse K63Ub of Tat, thereby preventing its destruction by autophagy (XREF_FIG).
Furthermore, TNFα and phospho-P65 was decreased while the level of IκBα was increased in the BRCC3 group, which confirmed that the abolition of BRCC3 inhibited the NF-κB signaling pathway (Figure 6E).
Here, we showed that most RNF213- and BRCC3 associated miRNAs were elevated in serum of MMD patients, repressed RNF213 and BRCC3 protein expression in HBMVEC, and modulated HBMVEC angiogenesis.
Morpholino mediated knockdown of the BRCC3 ortholog in zebrafish led to defective angiogenesis that was rescued by endothelial specific expression of brcc3, strongly suggesting that this deubiquitinat[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
For example, BRCC36 functions noncatalytically by recruiting USP13 to counteract the SMURF1-mediated degradation of STAT1, and this effect enhances the stability of STAT1 and improves host antiviral efficiency (110).
More specifically, BRCC36 deficiency leads to a rapid downregulation of STAT1 during viral infection, whereas complementation by BRCC36 can rescue the STAT1 expression levels and suppress virus infection (110).
However, in BRCC36 silenced cells, a large proportion of NuMA aggregates was retained in the peripheral areas distant from the spindle poles (XREF_FIG; 25-30 min), and supernumerary poles were formed, indicating that depletion of BRCC36 decreases the incorporation of NuMA into spindle poles during mitosis, presumably by aberrant dynamic interaction between NuMA and its partner dynein.
Immunoprecipitation revealed that depletion of ABRO1 or BRCC36 increased the interaction of NuMA with dynein, as well as with importin-beta in HeLa cells (XREF_FIG), indicating that BRISCs negatively regulate the association of NuMA with dynein and importin-beta.
BRCC3 gene knockdown through lentivirus mediated gene knockdown approach not only significantly reduced the clonogenic and migratory abilities of U251 and A172 cells, but also enhanced their sensitization to TMZ.
Our data showed that BRCC3 gene knockdown reduced the cell viability of TMZ treated U251 and A172 cells when compared to the relative TMZ treated mock cells.
Here, we showed that most RNF213- and BRCC3 associated miRNAs were elevated in serum of MMD patients, repressed RNF213 and BRCC3 protein expression in HBMVEC, and modulated HBMVEC angiogenesis.
Cytosolic SHMT2 was known to direct the BRCC36 containing complex (BRISC, a complex with deubiquitination activity) to deubiquitinate type I IFN receptor chain 1 (IFNalphaR1) and thus decrease the internalization and increase the stability of IFNalphaR1.
Interestingly, we revealed that BRCC36 knockdown decreased beta-catenin expression in CRC cells, suggesting that binding between SHMT2 and BRCC36 may direct BRCC36 activity towards K63-Ub chains conjugated to beta-catenin in CRC cells.
XREF_BIBR Importantly co-depletion of POH1 and BRCC36 did not elicit 53BP1 foci larger than POH1 depletion alone, suggesting these DUBs act in the same mechanistic pathway to restrict 53BP1 assemblies.
Furthermore, BRCC3 interference inhibited the cell viability, invasion and migration abilities of HeLa and SiHa cells via regulation of EMT progression and expression levels of Snai family members.
But beside the canonical role of deubiquitinating enzymes in antagonizing ubiquitin ligase activity, BRCC36 also enhances BRCA1 and BARD1 E3 ubiquitin ligase activity.
HSV is particularly effective in this antagonism, with HSV ICP0 promoting degradation of the host deubiquitinase BRCC36, resulting in downregulation of IFNAR1 in a variety of human and mouse cell lines in vitro [140] .
Although the suppressive role of BRCC36 in RNF8 dependent DDR suggests that BRCC36 promotes HR, the depletion of BRCC36 increases HR efficiency [XREF_BIBR].
Interestingly, the depletion of BRCC36 increases HR, which goes against the suppressive role of BRCC36 in RNF8 dependent DDR that should suggest that BRCC36 promotes HR.
In this complex, BRCC36 works together with the proteins NBA1, ABRO1, ABRA1 and BRCC45 in cleaving K63 linked ubiquitin chains and therefore seems to antagonize the ubiquitinating function of UBC13, RNF8 and RNF168.
In this complex, BRCC36 works together with the proteins NBA1, ABRO1, ABRA1 and BRCC45 in cleaving K63 linked ubiquitin chains and therefore seems to antagonize the ubiquitinating function of UBC13, RNF8 and RNF168.
But beside the canonical role of deubiquitinating enzymes in antagonizing ubiquitin ligase activity, BRCC36 also enhances BRCA1 and BARD1 E3 ubiquitin ligase activity.
This likely reflects the fact that the mechanism by which KIAA0157 and Abraxas support BRCC36 will differ from how CSN6 and RPN8 support and regulate the function of CSN5 and RPN11.
Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome, Associated-molecule-with-the-SH3-Domain-of-STAM (AMSH), which regulates ubiquitin dependent sorting of cell-surface receptors, and Brcc36, a K63 specific deubiquitnase of BRCC36 containing isopeptidase complex (BRISC) and BRCA1, BRCA2, and containing complex (BRCC).
Overexpressing SYVN1 promoted the ubiquitination and degradation of BRCC3 and reduced the secretion of pro-inflammatory cytokines in HG-induced Müller cells.
In addition, Real-time PCR analyses indicated that the ablation of BRCC3 reduced the expression of the NF-κB signaling down-stream genes including cIAP2, TNFα, and ICAM (Figure 4F).
We found that the downregulation of BRCC3 led to a specific and strong blockage of IL-1beta release triggered by ATP, while it had no effect on the NLRP3 independent TNF-alpha and IL-6 secretion induc[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
While truncation of BARD1 did not affect the association of any of the components of the complex, BRCA1 truncation completely abrogated the association of BRCC36 and BRE and reduced the association of[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The experiments were performed in triplicates comparing the cells treated with siRNAs against BRCC36 or BRE, to those treated with siRNAs against BRCA1 and control siRNAs.
We further identified that the ROS overproduction and mtDNA oxidative damage with upregulated deubiquitinase BRCC36, a potential molecular mechanism by which ocular surface epithelium initiates innate immune response to the environmental stress.