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 OTUD5 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
Two close relatives of DUBA, OTUB1 and OTUB2, were shown to reduce TRAF3 ubiquitination coupled with decreased anti-viral signaling responses in Sendai virus infected HEK 293T cells.
TRIAD3A dependent TRAF3 proteasomal destruction, like TRAF3 de-ubiquitination by DUBA, halts the activation of TBK1 and IKKepsilon and subsequent IFN production.
Interestingly, the newly identified cellular DUB, DUBA, inhibits the ubiquitination of TRAF3, an adaptor of RIG-I signaling, and negatively regulates the type I IFN signaling pathway 21.
Cellular protein DUBA specifically deubiquitinates TRAF3 that is critical for activation of IRF3, resulting in inhibiting phosphorylation of IRF3 (Kayagaki et al., 2007; Wang et al., 2011).
To verify that the deubiquitination of p53 by OTUD5 is specific, MDM2 was also tested in the in vitro deubiquitination assay and found thatMDM2 can not be deubiquitinated by OTUD5 (XREF_FIG).
We show that (1) OTUD5 is a novel p53 interacting protein; (2) OTUD5 deubiquitinates p53, leading to the stabilization of p53; (3) OTUD5 can help in the stabilization and activation of p53 in response to DNA damage; (4) OTUD5 is required for p53 induced apoptosis in response to DNA damage.
When DUBA deubiquitylates UBR5 and rescues it from degradation, UBR5 is free to polyubiquitylate RORgammaT, driving its degradation and consequently limiting Th17 differentiation.
DUBA interacted with the ubiquitin ligase UBR5, which suppressed DUBA abundance in naive T cells. DUBA accumulated in activated T cells and stabilized UBR5, which then ubiquitylated ROR纬t in response to TGF-尾 signalling.?
Given that TRAF3 may be dispensable for RIG-I signalling?92, it cannot be excluded, however, that DUBA also targets other E3 ligases like TRAF2, 5 and 6.
TRIAD3A dependent TRAF3 proteasomal destruction, like TRAF3 de-ubiquitination by DUBA, halts the activation of TBK1 and IKKepsilon and subsequent IFN production.
Given that TRAF3 may be dispensable for RIG-I signalling?92, it cannot be excluded, however, that DUBA also targets other E3 ligases like TRAF2, 5 and 6.
Given that TRAF3 may be dispensable for RIG-I signalling?92, it cannot be excluded, however, that DUBA also targets other E3 ligases like TRAF2, 5 and 6.
TRIAD3A dependent TRAF3 proteasomal destruction, like TRAF3 de-ubiquitination by DUBA, halts the activation of TBK1 and IKKepsilon and subsequent IFN production.
The function of OTUD5 is required to allow the rapid activation of p53 dependent transcription and a p53 dependent apoptosis in response to DNA damage stress.
It has been demonstrated that the deubiquitinating enzymes A20 and CYLD inhibit NF-kappaB signaling by targeting TRAF6 upstream of IKK, while the deubiquitinating protein DUBA inhibits type I interferon activity by targeting TRAF3.
OTUD5 has been shown to suppress the type I interferon dependent innate immune response by cleaving the polyubiquitin chain from an essential type I interferon adaptor protein.
For example, DUBA, a member of the Otubain (OTUB) family, has been shown to deubiquitinate TRAF3 and negatively regulate TLR3- and RIG-I and MDA5-mediated IFN induction.
For example, DUBA, a member of the Otubain (OTUB) family, has been shown to deubiquitinate TRAF3 and negatively regulate TLR3- and RIG-I/MDA5-mediated IFN induction (52).
The activation of TLR4 and TLR3 stimulates the K 63 -linked ubiquitination of TRAF3, which is required for the activation of IRF3 and the production of type I IFNs, and DUBA removes K 63 -linked ubiquitin chains from TRAF3, thereby inhibiting type I IFN responses.
Indeed, DUBA and OTUD5, a DUB which selectively disassembles Lys63 linked ubiquitin chains on TRAF3, has been shown to suppress the production of type I interferon by reducing the level of TRAF3-TBK1 interaction XREF_BIBR.
Structural data suggest that 1) Toll and IL-1R (TIR) domain containing regulators (BCAP, SIGIRR, and ST2) interfere with TIR domain signalosome formation; 2) major deubiquitinases such as A20, CYLD, and DUBA prevent association of TRAF6 and TRAF3 with their partners, inaddition to removing K63 linked ubiquitin chains that serve as a docking platform for downstream effectors; 3) alternative downstream pathways of TLRs also restrict signaling by competing to bind common partners through shared binding sites.
Moreover, the adaptor proteins TRAF3 and TRAF6 are targeted by the cellular proteins DUBA and A20 XREF_BIBR, XREF_BIBR, and TBK1 is sequestered by SIKE XREF_BIBR.
DUBA cleaves the K63-linked polyubiquitin chains of TRAF3 and depletion of DUBA increases the level of ubiquinated TRAF3 both in steady-state cells and in ligands-stimulated cells.
It has been reported that OTUD5 could suppress the type I interferon dependent innate immune response by cleaving the polyubiquitin chain from an essential type I interferon adaptor protein TRAF3.
OTUD5 has been shown to suppress the type I interferon dependent innate immune response by cleaving the polyubiquitin chain from an essential type I interferon adaptor protein.
Three human DUBs, CYLD, A20 and DUBA, have been shown to negatively regulate the innate immune response by removing K63-based chains [55, 56] , and USP15 inhibits the NFkB pathway by removing K48-Ub from IkBa, preventing its degradation [57] .
DUBA, a member of the ovarian tumor (OTU) domain containing cysteine protease superfamily, was shown to suppress the type I IFN dependent innate immune response by cleaving the K63 polyubiquitin chain on TRAF3.
Three human DUBs, CYLD, A20 and DUBA, have been shown to negatively regulate the innate immune response by removing K63 based chains XREF_BIBR, XREF_BIBR, and USP15 inhibits the NFkappaB pathway by removing K48-Ub from IkappaBalpha, preventing its degradation XREF_BIBR.
Three human DUBs, CYLD, A20 and DUBA, have been shown to negatively regulate the innate immune response by removing K63-based chains [55], [56], and USP15 inhibits the NFκB pathway by removing K48-Ub from IκBα, preventing its degradation [57].
The function of OTUD5 is required to allow the rapid activation of p53 dependent transcription and a p53 dependent apoptosis in response to DNA damage stress.
Knockdown of OTUD5 accelerated the proliferation , migration , and invasion of A549 cells , and inhibited their apoptosis Next , A549 cells were used for the following experiment .
Removal of Lys 63 -linked polyubiquitin chains from RIG-I and TRAF3 by cylindromatosis (CYLD) and deubiquitinating enzyme A (DUBA), respectively, inhibits the RIG-I-dependent activation of IRF3.
T-cell specific loss of DUBA therefore results in reduced levels of UBR5, stabilization of RORgammaT, and increased IL-17A production in response to TCR stimulation 76.
Accumulation of the deubiquitinating enzyme DUBA (OTUD5) negatively regulates RORgammat stability and IL-17 expression in T cells by stabilizing the ubiquitin ligase UBR5, which ubiquitinates RORgammat in response to TGF-beta, leading to RORgammat degradation and decreased IL-17 expression.
As DUBA protein is also increased in macrophages by LPS stimulation, it may also function as part of a negative feedback circuit.Another OTU DUB family protein, the tumor suppressor, CYLD, has been identified as a negative regulator of RIG-I by two groups [61, 62] .
For example, DUBA, a member of the Otubain (OTUB) family, has been shown to deubiquitinate TRAF3 and negatively regulate TLR3- and RIG-I and MDA5-mediated IFN induction.
For example, DUBA, a member of the Otubain (OTUB) family, has been shown to deubiquitinate TRAF3 and negatively regulate TLR3- and RIG-I/MDA5-mediated IFN induction (52).
Thus, OTUD5 controls neural cell fate commitment by regulating chromatin accessibility at neural- and neural crest specific enhancers to enable activation of transcriptional networks that drive the differentiation program.
Intriguingly, this separation-of-function mutant (OTUD5 DeltaCterm) failed to support neural crest differentiation and showed aberrant CNS precursor formation (XREF_FIG and fig.
Indeed, knockout of Otud5 is embryonic lethal in mice and OTUD5 depleted hESCs are defective in neuroectodermal differentiation, which can be rescued by re-expression of wild-type OTUD5 [XREF_BIBR].
OTUD5 expression was enhanced by IFN-gamma through a p38 / MAPK-dependent mechanism and the AS-induced knockdown of OTUD5 in LPMCs of IBD patients and colitic mice reduced TNF-alpha .
Recently, OTUD5 was found to be up-regulated in the intestinal inflammatory tissues of IBD patients and TNBS-induced colitis mice, and IFN-γ was found to up-regulate OTUD5’s expression through a p38/MAPK-dependent mechanism.
OTUD5 expression was enhanced by IFN-gamma through a p38 and MAPK dependent mechanism and the AS induced knockdown of OTUD5 in LPMCs of IBD patients and colitic mice reduced TNF-alpha.
As shown in XREF_FIG after Doxorubicin treatment depletion of OTUD5 clearly prevented the efficient deubiquitination of p53 that is associated with protein accumulation (XREF_FIG).
Therefore , it was concluded that OTUD5 could impede the proliferation , migration , and invasion of HCC827 cells while facilitating their apoptosis , and the role of OTUD5 was partly mediated by PDCD5 .
For example, DUBA, a member of the Otubain (OTUB) family, has been shown to deubiquitinate TRAF3 and negatively regulate TLR3- and RIG-I and MDA5-mediated IFN induction.
For example, DUBA, a member of the Otubain (OTUB) family, has been shown to deubiquitinate TRAF3 and negatively regulate TLR3- and RIG-I/MDA5-mediated IFN induction (52).
UBR5 destabilizes DUBA through ubiquitylation, whereas DUBA stabilizes UBR5 in activated T cells by attenuating degradative auto-ubiquitylation, triggering UBR5 mediated ubiquitylation of the transcription factor RORyt in response to TGF-beta [XREF_BIBR].
Structural data suggest that 1) Toll and IL-1R (TIR) domain containing regulators (BCAP, SIGIRR, and ST2) interfere with TIR domain signalosome formation; 2) major deubiquitinases such as A20, CYLD, and DUBA prevent association of TRAF6 and TRAF3 with their partners, inaddition to removing K63 linked ubiquitin chains that serve as a docking platform for downstream effectors; 3) alternative downstream pathways of TLRs also restrict signaling by competing to bind common partners through shared binding sites.
Moreover, the adaptor proteins TRAF3 and TRAF6 are targeted by the cellular proteins DUBA and A20 XREF_BIBR, XREF_BIBR, and TBK1 is sequestered by SIKE XREF_BIBR.
An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity.
Meylan and colleagues have shown that RIP3 inhibits RIP1 by competition, down-regulating the TRIF–RIP1-induced NF-κB pathway.40 Similarly to DUBA, A20 is a de-ubiquitinating enzyme that has been found to act in the cytoplasm as a negative regulator of TLR responses, affecting RIP1 and TRAF6 and thereby terminating TLR-induced NF-κB signalling.
The function of OTUD5 is required to allow the rapid activation of p53 dependent transcription and a p53 dependent apoptosis in response to DNA damage stress.
Interferon type I (IFN-I) responses are triggered by pattern-recognition receptors (PRRs), and DUBA (OTUD5) was shown to negatively regulate this immune signaling pathway [29] .
Two different lung tumor cell lines , H1299 and A549 , were used to investigate whether siRNA-mediated depletion of OTUD5 also promoted cell proliferation .
An intricate web of interactions involving the phosphate and the C-terminal tail of ubiquitin cause DUBA to fold around its substrate, revealing why phosphorylation is essential for deubiquitinase activity.
Further investigation reveals that OTUD5 depletion leads to the enhanced transcriptional activity of TRIM25 and the inhibited expression of PML by altering the ubiquitination level of TRIM25.
OTUD5 expression was enhanced by IFN-gamma through a p38 and MAPK dependent mechanism and the AS induced knockdown of OTUD5 in LPMCs of IBD patients and colitic mice reduced TNF-alpha.
Further investigation reveals that OTUD5 depletion leads to the enhanced transcriptional activity of TRIM25 and the inhibited expression of PML by altering the ubiquitination level of TRIM25.
DUBA knockdown by siRNA transfection in Il1r1 -/- BMDCs (XREF_FIG) resulted in significantly increased IL-10 and IFN-beta production and decreased secretion of IL-6, but not TNF, in CpG stimulated Il1r1 -/- BMDCs (XREF_FIG).
DUBA knockdown by siRNA transfection in Il1r1 -/- BMDCs (XREF_FIG) resulted in significantly increased IL-10 and IFN-beta production and decreased secretion of IL-6, but not TNF, in CpG stimulated Il1r1 -/- BMDCs (XREF_FIG).
To corroborate these results and exclude any off-target effects originating from clonal selection during iPSC reprogramming, we next generated control or OTUD5 depleted hES H1 cells and subjected them to neural conversion.
Given that TRAF3 may be dispensable for RIG-I signalling XREF_BIBR, it can not be excluded, however, that DUBA also targets other E3 ligases like TRAF2, 5 and 6.
In order to study whether OTUD5 can inhibit NSCLC via regulating p53 expression , OTUD5 siRNA and a p53 overexpressing plasmid were used to construct an si-OTUD5 / p53 group , an si-OTUD5 group , a p53 group , and an si-NC group , respectively .
Our findings reveal a mechanism whereby OTUD5 regulates gene transcription and suppresses tumorigenesis by deubiquitinating TRIM25 , providing a potential target for oncotherapy .
Here, we found that OTU deubiquitinase 5 (OTUD5) was bound to PDCD5 in response to etoposide treatment and increased the stability of PDCD5 by mediating deubiquitination of PDCD5 at Lys-97/98.
Several mechanisms dampen IFNα/β production, for example, deubiquitinating enzyme A (DUBA) is a negative regulator of IFNα/β downstream of multiple induction pathways [7].
DUBA knockdown by siRNA transfection in Il1r1 -/- BMDCs (XREF_FIG) resulted in significantly increased IL-10 and IFN-beta production and decreased secretion of IL-6, but not TNF, in CpG stimulated Il1r1 -/- BMDCs (XREF_FIG).
In other systems, IL-1 signaling has been shown to trigger the downregulation of deubiquitinating enzyme A (DUBA), which selectively cleaves K63 linked ubiquitin chains from TRAF3 to limit type I IFN responses.