OTUB1 Data Analysis

HGNC Gene Name
OTU deubiquitinase, ubiquitin aldehyde binding 1
HGNC Gene Symbol
OTUB1
Identifiers
hgnc:23077 NCBIGene:55611 uniprot:Q96FW1
Orthologs
mgi:2147616 rgd:1311329
INDRA Statements
deubiquitinations all statements
Pathway Commons
Search for OTUB1
Number of Papers
147 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
UBE2D3 ubiquitin conjugating enzyme E2 D3 0.351 BioGRID IntAct INDRA (6) Reactome (2)
UBE2D2 ubiquitin conjugating enzyme E2 D2 0.259 BioGRID IntAct INDRA (5) Reactome (2) 0.43 2.28 2.33e-14
UBR4 ubiquitin protein ligase E3 component n-recognin 4 0.218 0.49 2.63 1.50e-22
BTG1 BTG anti-proliferation factor 1 0.216
MDM4 MDM4 regulator of p53 0.212 INDRA (3) Reactome (4)
MS4A10 membrane spanning 4-domains A10 0.209
UBE2C ubiquitin conjugating enzyme E2 C 0.189 Reactome (2) 0.25 1.30 7.36e-06

Dependency GO Term Enrichment

Gene set enrichment analysis was done on the genes correlated with OTUB1using 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
GO:0070979 protein K11-linked ubiquitination Biological Process 7.11e-08 8.60e-06 3.29e-06
GO:0061650 ubiquitin-like protein conjugating enzyme activity Molecular Function 1.73e-07 2.09e-05 4.00e-06
GO:0070936 protein K48-linked ubiquitination Biological Process 4.58e-07 5.54e-05 7.07e-06
GO:0019787 ubiquitin-like protein transferase activity Molecular Function 3.05e-06 3.70e-04 3.54e-05
GO:0002756 MyD88-independent toll-like receptor signaling pathway Biological Process 4.12e-05 4.98e-03 3.81e-04
GO:0000151 ubiquitin ligase complex Cellular Component 6.22e-05 7.52e-03 4.78e-04
GO:0044389 ubiquitin-like protein ligase binding Molecular Function 8.01e-05 9.70e-03 4.78e-04
GO:0000209 protein polyubiquitination Biological Process 8.25e-05 9.98e-03 4.78e-04
GO:0051865 protein autoubiquitination Biological Process 1.88e-04 2.28e-02 9.23e-04
GO:0043574 peroxisomal transport Biological Process 1.99e-04 2.41e-02 9.23e-04
GO:0007031 peroxisome organization Biological Process 2.67e-04 3.23e-02 1.12e-03

Literature Mining

INDRA was used to automatically assemble known mechanisms related to OTUB1 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
OTUB1 deubiquitinates TRAF3. 7 / 7
| 6

reach
OTUB1 and OTUB2 interact with and deubiquitinate TRAF3 and TRAF6, which are required for virus triggered IRF3 and NF-kappaB activation [XREF_BIBR].

ubibrowser
Regulation of virus-triggered signaling by OTUB1- and OTUB2-mediated deubiquitination of TRAF3 and TRAF6

reach
It was also shown that two OTUB deubiquitinating enzyme family members, OTUB1 and OTUB2, can deubiquitinate TRAF3 and TRAF6, leading to the inhibition of virus induced IFN-beta expression and cellular antiviral responses.

reach
Knockout of HSCARG attenuated the de-ubiquitination of TRAF3 by OTUB1, and knockdown of OTUB1 abolished the effect of HSCARG.

reach
OTUB1 and its paralog OTUB2, deubiquitinate TRAF3 and TRAF6 to inhibit virus triggered signaling pathways that ultimately result in IRF3 and NF-kappaB activation [XREF_BIBR].

reach
Furthermore, we found that OTUB1 and OTUB2 mediated virus triggered deubiquitination of TRAF3 and -6.

reach
It was also shown that two OTUB deubiquitinating enzyme family members, OTUB1 and OTUB2, can deubiquitinate TRAF3 and TRAF6, leading to the inhibition of virus-induced IFN-β expression and cellular antiviral responses (53).
OTUB1 leads to the deubiquitination of TP53. 7 / 7
| 6

reach
Otub1, a DUB from the OTU-domain containing protease family abrogates p53 ubiquitination and stabilizes and activates p53 in cells independent of its deubiquitinating enzyme activity [XREF_BIBR].

reach
OTUB1 blocks p53 ubiquitylation by MDM2 by interacting with and inhibiting UbcH5, and E2 conjugating enzyme for MDM2.

ubibrowser
Review

reach
Examples for DUBs that might antagonize E4 dependent polyubiquitylation are USP47, a regulator of Base Excision Repair (BER) that controls DNA polymerase beta and OTUB1, which mediates DNA damage dependent deubiquitylation of p53 and MDM2 in the cytoplasm.

reach
Interestingly, Otub1 abrogates p53 ubiquitination and stabilizes and activates p53 in cells independently of its deubiquitinating enzyme activity.

reach
For example, USP7 regulates the stability of both p53 and Mdm2 and maintains p53 ubiquitination levels; 120 USP2 mediates the stability of Mdm2; 121 USP10 modulates p53 localization and stability; 122 OTUB1 abrogates p53 ubiquitination and activates p53.

reach
For example, USP7 modulates the stability of both p53 and MDM2, and maintains the level of p53 ubiquitylation; USP2 affects the stability of MDM2; Otub1 inhibits p53 ubiquitination and activates p53 in cells; USP10 regulates the location and stability of p53, and stabilize both mutated and wild-type p53, thereby having a dual role in tumorigenesis.
OTUB1 leads to the deubiquitination of RAS. 6 / 6
| 6

reach
OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane.

reach
Consistent with the observation that OTUB1 inhibits RAS ubiquitination, analysis of RAS localization revealed that OTUB1 overexpression augmented the presence of RAS proteins on the plasma membrane (Fig XREF_FIG E and F; XREF_SUPPLEMENTARY).

reach
OTUB1 triggers activation of the MAPK pathway by inhibiting RAS ubiquitination.

reach
Therefore, we tested whether the catalytic activity of OTUB1 is essential to promote RAS de-ubiquitination.

reach
In fact, invitro ubiquitination of RAS was abolished by wt OTUB1, but not by deltaN (1-30) OTUB1-mutant lacking binding to E2 (Fig XREF_FIG A).

reach
OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination.
OTUB1 deubiquitinates FOXM1. 5 / 5
| 4

reach
29 We have shown previously that OTUB1 can specifically limit FOXM1 polyubiquitination and its degradation, through its deubiquitinase enzymatic activity.

reach
Conversely, the invitro deubiquitination assays revealed that both OTUB1 (WT) and OTUB1 (D88A), but not OTUB1 (C91A), could efficiently deubiquitinate FOXM1 (XREF_FIG C, invitro Deub, lanes 5-8), as expected.

reach
Taken together, the results provided strong evidence that OTUB1 suppresses FOXM1 ubiquitination and degraduation.

ubibrowser
OTUB1-catalyzed deubiquitination of FOXM1 facilitates tumor progression and predicts a poor prognosis in ovarian cancer

reach
Moreover, OTUB1 can deubiquitinate and stabilize the forkhead transcription factor FOXM1, which promotes DNA damage response and genotoxic drug resistance in breast cancer.
OTUB1 leads to the deubiquitination of MSH2. 5 / 5
| 5

reach
These results strongly suggest that OTUB1 blocks MSH2 ubiquitination by inhibiting the E2 ligase activity, rather than by directly deubiquitinating MSH2.

reach
OTUB1 inhibits the ubiquitination of MSH2.

reach
It is possible that OTUB1 prevents MSH2 ubiquitination by inhibiting the E2 activity of HDAC6 or by competing with HDAC6 for the MSH2 residues located in the central domain.

reach
Collectively, these results suggest that MSH2 is a substrate of OTUB1 and that OTUB1 inhibits MSH2 ubiquitination.

reach
We therefore conclude that OTUB1 prevents MSH2 ubiquitination by suppressing the E2 ligase activity.
OTUB1 deubiquitinates TRAF6. 5 / 5
| 4

reach
It was also shown that two OTUB deubiquitinating enzyme family members, OTUB1 and OTUB2, can deubiquitinate TRAF3 and TRAF6, leading to the inhibition of virus-induced IFN-β expression and cellular antiviral responses (53).

reach
OTUB1 and OTUB2 interact with and deubiquitinate TRAF3 and TRAF6, which are required for virus triggered IRF3 and NF-kappaB activation [XREF_BIBR].

ubibrowser
Regulation of virus-triggered signaling by OTUB1- and OTUB2-mediated deubiquitination of TRAF3 and TRAF6

reach
It was also shown that two OTUB deubiquitinating enzyme family members, OTUB1 and OTUB2, can deubiquitinate TRAF3 and TRAF6, leading to the inhibition of virus induced IFN-beta expression and cellular antiviral responses.

reach
OTUB1 and its paralog OTUB2, deubiquitinate TRAF3 and TRAF6 to inhibit virus triggered signaling pathways that ultimately result in IRF3 and NF-kappaB activation [XREF_BIBR].
OTUB1 leads to the deubiquitination of RNF168. 4 / 4
| 4

reach
Indeed, the ability of OTUB1 to inhibit RNF168 dependent ubiquitylation is independent of its catalytic activity, and is instead mediated by its binding to and inhibition of the E2 UBC13, which cooperates with RNF168, suggesting that E2 regulation could also represent a means to regulate the DDR pathway.

reach
Finally, the DUB OTUB1 inhibits RNF168 mediated ubiquitylation in a non catalytic fashion by binding to UBC13 and inhibiting the formation of Lys63 linked poly-ubiquitin chains [35 **].

reach
It has been reported that OTUB1 suppresses RNF168 dependent poly-ubiquitination by binding to and inhibiting UBC13, but independently of OTUB1 catalytic activity as a deubiquitinating enzyme (Nakada e[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

reach
Remarkably, the ability of OTUB1 to inhibit RNF168 dependent ubiquitylation was independent of its catalytic activity, suggesting an unusual mechanism of action.
OTUB1 leads to the deubiquitination of UBE2E1. 4 / 4
| 2

ubibrowser
OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

reach
A recent study showed that OTUB1 also prevents autoubiquitylation of UBE2E1, thereby rescuing the E2 from proteasomal degradation 35.

ubibrowser
We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines.

reach
OTUB1 inhibits auto-monoubiquitylation of UBE2E1 and UBE2E2 but not UBE2E3.
OTUB1 deubiquitinates SMAD3. 3 / 3
| 2

reach
OTUB1 could not directly de-ubiquitinate Smad2 and Smad3, while its effect is dependent on regulating ubiquitin E2 conjugating-enzyme activity.

reach
OTUB1 prevents SMAD3 ubiquitylation.

ubibrowser
OTUB1 enhances TGFbeta signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.
OTUB1 deubiquitinates SMAD2. 3 / 3
| 2

ubibrowser
OTUB1 enhances TGFbeta signalling by inhibiting the ubiquitylation and degradation of active SMAD2/3.

reach
OTUB1 could not directly de-ubiquitinate Smad2 and Smad3, while its effect is dependent on regulating ubiquitin E2 conjugating-enzyme activity.

reach
Recently, OTUB1, a DUB, was reported to reduce poly-ubiquitination of Smad2 and Smad3 through inhibition of the E2 ubiquitin conjugating enzymes.
OTUB1 deubiquitinates SMAD2_3. 3 / 3
| 3

reach
These results imply that OTUB1 inhibits the ubiquitylation of SMAD2/3 rather than directly deubiquitylating them.

reach
Interestingly, it was found that the mechanism is non canonical and non catalytic : instead of OTUB1 directly deubiquitinating SMAD2/3 rather it interacts with the E2 ligases prevents the transfer of [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

reach
This led us to hypothesize that OTUB1 might inhibit the ubiquitylation of SMAD2/3.
OTUB1 leads to the deubiquitination of UBE2N. 2 / 2
| 1

reach
Since, OTUB1 inhibits UBC13 dependent polyubiquitylation but not monoubiquitylation, it may be that a single ubiquitin conjugated to another protein at DSBs is sufficient to be recognized by the MIU d[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

ubibrowser
Although OTUB1 is not catalytically involved in deubiquitinating these K63-linked chains, it may interact with Ubc13 and inhibit the E3 ligase RNF168
OTUB1 deubiquitinates MAPT. 2 / 2
| 1

ubibrowser
Focusing on Tau deubiquitination, we here identify Otub1 as a Tau-deubiquitinating enzyme. Otub1 directly affected Lys48-linked Tau deubiquitination

reach
We demonstrate here for the first time that Tau is deubiquitinated by Otub1.
OTUB1 deubiquitinates ESR1. 2 / 2
| 1

reach
OTUB1 deubiquitinates ERalpha in vitro and in vivo and stabilizes ERalpha in the nucleus [XREF_BIBR].

ubibrowser
No evidence text available
OTUB1 leads to the deubiquitination of DDX58. 2 / 2
| 2

reach
NS1 is the most important IFNs antagonist protein via mechanisms including inhibition of the TRIM25 mediated RIG-I ubiquitination, suppression of protein kinase R (PKR), phosphorylation of IkappaB kinases (IKK) alpha and beta in the NF-kappaB pathway, interruption of the phosphorylation of STAT1, STAT2, and STAT3 [XREF_BIBR, XREF_BIBR], and degradation of OTUB1 [XREF_BIBR].

reach
NS1 is the most important IFNs antagonist protein via mechanisms including inhibition of the TRIM25-mediated RIG-I ubiquitination, suppression of protein kinase R (PKR), phosphorylation of IκB kinases (IKK) α and β in the NF-κB pathway, interruption of the phosphorylation of STAT1, STAT2, and STAT3 [39, 115] , and degradation of OTUB1 [138] .
OTUB1 deubiquitinates SNAI1. 1 / 1
|

ubibrowser
Functionally, OTUB1 facilitates metastasis of esophageal squamous cell carcinoma (ESCC) through promoting Snail protein stability.
Ubiquitinated OTUB1 leads to the deubiquitination of SMAD3. 1 / 1
| 1

reach
In contrast, OTU domain, ubiquitin aldehyde binding 1 (OTUB1), B-cell lymphoma-3 (BCL-3), ubiquitin carboxyl-terminal hydrolase 1 (UCHL1), and UCHL5 contribute to the deubiquitination of SMAD2 or SMAD3, making them more stable and less easily degradable and promoting TGF-beta signaling [XREF_BIBR - XREF_BIBR].
| PMC
OTUB1 deubiquitinates RPA1. 1 / 1
|

ubibrowser
PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination.
OTUB1 deubiquitinates DEPTOR. 1 / 1
|

ubibrowser
OTUB1 protein suppresses mTOR complex 1 (mTORC1) activity by deubiquitinating the mTORC1 inhibitor DEPTOR
Ubiquitinated OTUB1 leads to the deubiquitination of SMAD2. 1 / 1
| 1

reach
In contrast, OTU domain, ubiquitin aldehyde binding 1 (OTUB1), B-cell lymphoma-3 (BCL-3), ubiquitin carboxyl-terminal hydrolase 1 (UCHL1), and UCHL5 contribute to the deubiquitination of SMAD2 or SMAD3, making them more stable and less easily degradable and promoting TGF-beta signaling [XREF_BIBR - XREF_BIBR].
| PMC
OTUB1-C91S leads to the deubiquitination of Histone. 1 / 1
| 1

reach
However, the inhibitory effect of OTUB1 is independent of its DUB activity because the catalytically inactive mutant OTUB1 C91S suppresses 53BP1 IRIF and core histone ubiquitination as efficiently as wild-type OTUB1 [XREF_BIBR].
OTUB1-D88A deubiquitinates FOXM1. 1 / 1
| 1

reach
Conversely, the invitro deubiquitination assays revealed that both OTUB1 (WT) and OTUB1 (D88A), but not OTUB1 (C91A), could efficiently deubiquitinate FOXM1 (XREF_FIG C, invitro Deub, lanes 5-8), as expected.
OTUB1 deubiquitinates BIRC2. 1 / 1
|

ubibrowser
Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex.
OTUB1 deubiquitinates forkhead. 1 / 1
| 1

reach
Moreover, OTUB1 can deubiquitinate and stabilize the forkhead transcription factor FOXM1, which promotes DNA damage response and genotoxic drug resistance in breast cancer.
Mutated OTUB1 leads to the deubiquitination of SMAD2_3. 1 / 1
| 1

reach
Further, some catalytically inactive mutants of OTUB1, including OTUB1 C91S, were also able to inhibit SMAD2/3 ubiquitylation in vitro (XREF_FIG).
OTUB1 leads to the deubiquitination of UBE2E3. 1 / 1
| 1

reach
These results suggest that OTUB1 may serve to primarily inhibit polyubiquitination of UBE2E3, but not autoubiquitylation.
OTUB1 leads to the deubiquitination of TGFB. 1 / 1
| 1

reach
OTUB1, a member of the OTU family of DUBs, has been shown to inhibit the ubiquitylation of only TGFbeta activated SMAD2/3 XREF_BIBR.
Modified OTUB1 leads to the deubiquitination of DCN. 1 / 1
| 1

reach
The overexpression of OTUB1 enhanced the ubiquitination and degradation of DCN in MC.
OTUB1 leads to the deubiquitination of MDM2. 1 / 1
| 1

reach
Examples for DUBs that might antagonize E4 dependent polyubiquitylation are USP47, a regulator of Base Excision Repair (BER) that controls DNA polymerase beta and OTUB1, which mediates DNA damage dependent deubiquitylation of p53 and MDM2 in the cytoplasm.
OTUB1 deubiquitinates EYA1. 1 / 1
| 1

reach
Mechanistically, OTUB1 could interact with EYA1 directly and deubiquitinate EYA1 to stabilize it.
OTUB1 deubiquitinates FLAG. 1 / 1
| 1

reach
Wild-type OTUB1 and the DeltaN mutant, both capable of cleaving K48 linked di- or polyubiquitin chains in vitro, along with all the catalytically inactive mutants (D88A, C91S, H265A, D/H, D/C/H, K71R) were not able to deubiquitylate the polyubiquitylated FLAG and SMAD2/3/4 complex (XREF_FIG).
OTUB1 leads to the deubiquitination of E2F1. 1 / 1
| 1

reach
Furthermore, the OTUB1 knockdown significantly reversed the decreased ubiquitination of E2F1 induced by the overexpression of TMPO-AS1 in RT4 and T24 cells (XREF_FIG).
OTUB1-C91A deubiquitinates FOXM1. 1 / 1
| 1

reach
Conversely, the invitro deubiquitination assays revealed that both OTUB1 (WT) and OTUB1 (D88A), but not OTUB1 (C91A), could efficiently deubiquitinate FOXM1 (XREF_FIG C, invitro Deub, lanes 5-8), as expected.
Modified OTUB1 leads to the deubiquitination of Histone. 1 / 1
| 1

reach
Whereas the exogenous expression of OTUB1 completely abrogated RNF168 dependent histone ubiquitination, the exogenous expression of OTUB2 did not affect the histone ubiquitination level.

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
OTUB1 affects TP53
| 6 17
OTUB1 activates TP53.
| 6 12
OTUB1 activates TP53. 10 / 26
| 6 12

reach
Mitochondrial ROS scavengers prevented OTUB1-mediated p53 stabilization and Bax upregulation by ODN.

reach
This non canonical mode of regulation was also reported when OTUB1 was shown to stabilize and activate p53 independent of catalytic activity [XREF_BIBR].

reach
Overexpression of OTUB1 induces p53 dependent apoptosis whereas its knockdown attenuates p53 activation.

sparser
For example, USP7 modulates the stability of both p53 and MDM2, and maintains the level of p53 ubiquitylation ( xref , xref ); USP2 affects the stability of MDM2 ( xref ); Otub1 inhibits p53 ubiquitination and activates p53 in cells ( xref ); USP10 regulates the location and stability of p53, and stabilize both mutated and wild-type p53, thereby having a dual role in tumorigenesis ( xref ).

reach
Inhibition of cathepsin K sensitizes oxaliplatin-induced apoptotic cell death by Bax upregulation through OTUB1-mediated p53 stabilization in vitro and in vivo.

sparser
Interestingly, Otub1 abrogates p53 ubiquitination and stabilizes and activates p53 in cells independently of its deubiquitinating enzyme activity.

reach
Casein kinase 2 (CK2)-dependent phosphorylation of OTUB1 at Ser16 played a critical role in ODN- and cathepsin K siRNA-mediated p53 stabilization.

reach
Consequently, overexpression of Otub1 induces p53 dependent apoptosis and inhibition of cell proliferation whereas knockdown of Otub1 attenuates p53 activation following DNA damage [XREF_BIBR].

sparser
For example, USP7 regulates the stability of both p53 and Mdm2 and maintains p53 ubiquitination levels; xref USP2 mediates the stability of Mdm2; xref USP10 modulates p53 localization and stability; xref OTUB1 abrogates p53 ubiquitination and activates p53. xref Interestingly, USP10 can stabilize both mutated and wild-type p53, with a dual role in tumorigenesis.

reach
In summary, we have found that Otub1 promotes p53 function by stabilizing MDMX, which in turn contributes to the p53 induced mitochondria mediated apoptosis pathway.
OTUB1 inhibits TP53.
| 3
OTUB1 inhibits TP53. 2 / 4
| 2

reach
This is particularly interesting when considering our observations that in prostate cancer cells, OTUB1 mediates the androgen inhibition of p53 through a mechanism that requires an intact OTUB1 catalytic motif and seems to be independent from MDM2 concentration changes.

reach
Our results demonstrate that cathepsin K inhibition enhances oxaliplatin-induced apoptosis by increasing OTUB1 phosphorylation via CK2 activation, thereby promoting p53 stabilization, and hence upregulating Bax.
OTUB1-D88A inhibits TP53. 1 / 2
| 1

reach
Overexpression of Otub1 (D88A) or ablation of endogenous Otub1 by siRNA markedly impaired p53 stabilization and activation in response to DNA damage.
OTUB1 increases the amount of TP53.
| 2
OTUB1 increases the amount of TP53. 1 / 2
| 1

reach
These results suggest that Otub1 increases the cytoplasmic levels of both MDMX and p53.
Modified OTUB1 increases the amount of TP53. 1 / 1
| 1

reach
As shown in Figure XREF_FIG, cell fractionation assays revealed that Otub1 expression not only significantly induced the nuclear levels of p53 and MDMX, but also induced the levels of cytoplasmic MDMX and p53 (lane 4, Figure XREF_FIG).
OTUB1 affects E2
| 1 4 28
OTUB1 inhibits E2.
| 1 4 25
OTUB1 inhibits E2. 10 / 29
| 1 4 24

reach
The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes in a non catalytic manner.

reach
On this basis, it has been proposed that OTUB1 inhibits the ability of E2 ~ Ub conjugates to participate in Ub transfer reactions and in building polyUb chains 2.

eidos
This E2 suppression by OTUB1 inhibits RNF168 mediated K63 polyubiquitylation in the DNA damage site .

reach
We next investigated whether OTUB1 inhibits E2 by preventing the conjugation of ubiquitin or the transfer of ubiquitin from E2 to E3.

reach
Further characterization of this mechanism demonstrated that OTUB1 directly binds and consequently inhibits a related subclass of E2 enzymes that include UBC13, the only known E2 that cooperates with RNF168 during the DNA damage response [23], [195].

reach
OTUB1 interacts with UBC13 (UBE2N) and the UBE2D and UBE2E family E2 ubiquitin conjugating enzymes and inhibits their E2 activities in a DUB activity independent manner (Nakada et al., 2010; Sato et a[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

reach
Intriguingly, OTUB1 inhibits UBC13 and UBE2D/2E family E2 conjugating enzymes in a DUB activity independent manner [XREF_BIBR].

reach
Occlusion of the E3 binding surface, along with the shielding of the Ub ~ UbcH5b ester linkage by OTUB1 likely accounts for the ability of OTUB1 to suppress E2 function.

reach
However, its inhibitory effect was higher than that of an OTUB1 N-terminal deletion mutant that does not inhibit the E2 activity of UBC13 (Nakada et al., 2010).

sparser
We next investigated whether OTUB1 inhibits E2 by preventing the conjugation of ubiquitin or the transfer of ubiquitin from E2 to E3.
OTUB1 bound to UBE2N inhibits E2. 1 / 1
| 1

reach
OTUB1 interacts with UBC13 (UBE2N) and the UBE2D and UBE2E family E2 ubiquitin conjugating enzymes and inhibits their E2 activities in a DUB activity independent manner (Nakada et al., 2010; Sato et a[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
OTUB1 activates E2.
| 3
OTUB1 activates E2. 3 / 3
| 3

reach
OTUB1 activates RIG-I via a dual mechanism ofK48 polyubiquitin hydrolysis and formation of an E2 and repressive complex with UBCH5c.

reach
Recently, several studies have described a non canonical mode of OTUB1 action in which OTUB1 inhibits the ubiquitylation of target proteins by binding to and inhibiting the E2 ubiquitin conjugating enzyme UBE2N (also known as UBC13) independent of its catalytic activity XREF_BIBR XREF_BIBR XREF_BIBR XREF_BIBR.

reach
This and other studies have reported that OTUB1 inhibits ubiquitylation by binding to and inhibiting E2 enzymes XREF_BIBR XREF_BIBR XREF_BIBR XREF_BIBR XREF_BIBR.
OTUB1 affects Ubiquitin
| 2 24
OTUB1 inhibits Ubiquitin.
| 2 18
| 2 15

reach
These results suggest that by binding to E2 ubiquitin conjugating enzymes, OTUB1 appears to inhibit the transfer of ubiquitin from E2-Ub complex onto E3 ubiquitin ligases.

reach
In contrast to BRCC36, USP16 and USP3, OTUB1 inhibits the DSB ubiquitin response downstream of RNF8, at the level of RNF168.

reach
IL-15 mediates membrane recruitment of Otub1, which inhibits ubiquitin dependent activation of AKT, a kinase that is pivotal for T cell activation and metabolism.

reach
In contrast to BRCC36, USP16 and USP3, OTUB1 inhibits the DSB ubiquitin response downstream of RNF8, at the level of RNF168.

reach
The OTUB1 protein interacts directly with the E2 ubiquitin conjugating protein UBC13 and prevents ubiquitin transfer, thereby inhibiting double-strand-break-induced chromatin ubiquitination XREF_BIBR, XREF_BIBR.

reach
We next investigated whether OTUB1 inhibits E2 by preventing the conjugation of ubiquitin or the transfer of ubiquitin from E2 to E3.

reach
This occurs by different molecular mechanisms and requires catalytic activity of USP15, but not that of OTUB1, which rather binds to and inhibits the ubiquitin conjugating activity of the cognate E2 enzyme [XREF_BIBR, XREF_BIBR].

reach
In addition to such enzymatic ubiquitin deconjugation, the DUB OTUB1 limits ubiquitin signaling by direct inhibition of the ubiquitin conjugating enzyme UBC13 [68-70], while the ubiquitin E3 ligase RN[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

reach
OTUB1 binds to a subset of E2 ubiquitin conjugating enzymes and inhibits their activity by trapping the E2 ~ ubiquitin thioester and preventing ubiquitin transfer.

reach
We further showed that OTUB1, by binding to UBE2D1, inhibits the transfer of ubiquitin from E2-Ub conjugate to E3 ubiquitin ligase NEDD4L and subsequently to SMAD3 (XREF_FIG).
OTUB1-K71R inhibits Ubiquitin. 1 / 1
| 1

reach
However, OTUB1 K71R, a catalytically inactive mutant, does not appear to inhibit the transfer of ubiquitin from the E2-Ub complex to E3 (XREF_FIG cf. bottom line mono-ubiquitin).
OTUB1 bound to E2 inhibits Ubiquitin. 1 / 1
| 1

reach
OTUB1 binds to E2 ~ Ub thioester intermediates and prevent ubiquitin transfer, thereby non catalytically inhibiting accumulation of polyubiquitin.
OTUB1 bound to UBE2D1 inhibits Ubiquitin. 1 / 1
| 1

reach
Further, UbcH5 in turn mediates Otub1 monoubiquitination and this monoubiquitination facilitates the Otub1 binding to UbcH5 and likely inhibits ubiquitin chain transfer [XREF_BIBR], providing a mechanism underlying the Otub1 inhibition of E2 activity.
OTUB1 activates Ubiquitin.
| 4
| 3

reach
OTUB1 has previously been reported to inhibit ubiquitylation of target proteins by interacting with and inhibiting E2 ubiquitin conjugating enzymes XREF_BIBR XREF_BIBR XREF_BIBR XREF_BIBR.

reach
Immunoblot analysis revealed that ubiquitin chains were completely degraded by AMSH but neither by OTUB1 nor the GST control, further supporting our finding that TRIAD3 synthesises K63 linked ubiquitin chains in vitro, full length TRIAD3 protein as well as the TRIAD3-RBR 845 fragment.

reach
For this, heterotypic Ub chains were assembled from 25 mg WT Ub, and the reaction was subsequently treated with OTUB1, which cleaved all Lys48-linkages within the polymers.
OTUB1 bound to E2 activates Ubiquitin. 1 / 1
| 1

reach
OTUB1 binds to a subset of E2 ubiquitin conjugating enzymes and inhibits their activity by trapping the E2 ~ ubiquitin thioester and preventing ubiquitin transfer.
OTUB1 decreases the amount of Ubiquitin.
| 2
Modified OTUB1 decreases the amount of Ubiquitin. 2 / 2
| 2

reach
In contrast, only overexpression of mammalian Otubain 1 and Cezanne moderately decreased cellular global Ub conjugate levels, while expression of A20 or Otubain 2 had no effect on total levels of ubiquitinated proteins (Balakirev et al., 2003; Evans et al., 2004; Evans et al., 2003) .

reach
In contrast, only overexpression of Otubain 1 and Cezanne moderately decreased cellular global Ub conjugate levels, while expression of A20 or Otubain 2 had no effect on total levels of ubiquitinated proteins (Balakirev et al., 2003, Evans et al., 2003, Evans et al., 2004).
OTUB1 affects UBE2N
1 | 8 15
OTUB1 inhibits UBE2N.
1 | 8 13
1 | 8 11

reach
Consistent with a functional importance of this pocket, the F190S mutation disrupted the ability of OTUB1 to suppress UBC13 activity in vitro (XREF_FIG).

reach
OTUB1 strongly suppresses UBC13 dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro.

sparser
OTUB1 inhibits the RNF168 E2 enzyme UBE2N (and probably also other E2s) independently of its de-ubiquitylating activity [ xref , xref ].

sparser
Another DUB, OTUB1, directly binds and inhibits the E2 Ubc13 by a mechanism independent of its catalytic isopeptidase activity [ xref ].

reach
Although human ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) was previously identified as a K48 linkage specific deubiquitinating enzyme that positively regulates p53 stability 22, OTUB1 strongly suppresses E2-ubiquitin-conjugating enzyme UBC13 dependent K63 linked ubiquitination XREF_BIBR, XREF_BIBR.

sparser
OTUB1 directly inhibits the E2 enzyme UBC13, independently of its catalytic activity.

reach
It is worth noting that OTUB1 inhibits UBE2D/2E family in in vitro experiments as well as UBC13, indicating that deubiquitylating activity independent regulation of E2 might be a common mechanism for [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

reach
It was reported that Otub1, a deubiquitinase, also negatively regulates the DDR in a catalytically independent manner by regulating the activity of UBC13, an E2 ubiquitin conjugating enzyme for RNF8.

sparser
Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes.

sparser
Intriguingly, OTUB1 inhibits UBC13 and UBE2D/2E-family E2 conjugating enzymes in a DUB activity-independent manner [ xref ].
OTUB1-D137G inhibits UBE2N. 1 / 1
| 1

reach
OTUB1 D137G also failed to inhibit UBC13 (XREF_FIG).
Mutated OTUB1 inhibits UBE2N. 1 / 1
| 1

reach
Finally, we carried out GST pull-down assays with the OTUB1 mutants that failed to inhibit UBC13 and found that all had reduced binding to Ub ~ UbcH5b and free Ub (XREF_SUPPLEMENTARY).
OTUB1 activates UBE2N.
| 2
OTUB1 activates UBE2N. 2 / 2
| 2

reach
The deubiquitinase OTUB1 augments NF-kappaB-dependent immune responses in dendritic cells in infection and inflammation by stabilizing UBC13.

reach
Interestingly, OTUB1 is not involved in the cleavage of polyubiquitin chains but directly targets UBC13 [77].
OTUB1 affects FOXM1
| 15
OTUB1 activates FOXM1.
| 5
OTUB1 activates FOXM1. 4 / 8
| 4

reach
This finding was interesting as OTUB1 inhibits the degradation of FOXM1 transcription factor, typically upregulated and overexpressed in aggressive therapy resistant breast cancer.

reach
OTUB1 enhances FOXM1 stability in response to epirubicin treatment.

reach
Our data show that WT OTUB1, but not the OTUB1 (C91S) catalytic dead mutant, can enhance the stability of FOXM1 in response to epirubicin in MCF-7 cells, suggesting that the deubiquitinase activity is required for the degradation of FOXM1.

reach
The fact that OTUB1 specifically targets FOXM1 also supports the fact that FOXM1 is ubiquitinated by K48-linkage chains.
OTUB1-C91S activates FOXM1. 1 / 1
| 1

reach
Our data show that WT OTUB1, but not the OTUB1 (C91S) catalytic dead mutant, can enhance the stability of FOXM1 in response to epirubicin in MCF-7 cells, suggesting that the deubiquitinase activity is required for the degradation of FOXM1.
OTUB1 inhibits FOXM1.
| 2
OTUB1 inhibits FOXM1. 2 / 7
| 2

reach
Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1 (C91S) mutant, significantly enhances the half-life of FOXM1.

reach
Together, these results suggest that the suppression of FOXM1 degradation by OTUB1 in response to epirubcin requires its deubiquitinating catalytic activity, further confirming that FOXM1 is a novel target of the deubiquitinase OTUB1.
OTUB1 increases the amount of FOXM1.
| 7
OTUB1 increases the amount of FOXM1. 5 / 5
| 5

reach
The results showed that in MCF-7 cells OTUB1 depletion culminated in the downregulation of FOXM1, but not vice versa (XREF_FIG), suggesting that OTUB1 positively regulates FOXM1 expression.

reach
To investigate the idea that OTUB1 modulates FOXM1 expression further, we examined the effects of silencing OTUB1 and FOXM1 on the expression of endogenous OTUB1 and FOXM1 protein in MCF-7 cells.

reach
Mechanistically, knockdown of OTUB1 down-regulated FOXM1 expression by promoting its ubiquitination.

reach
Given our previous finding that genotoxic agents cause FOXM1 SUMOylation, ubiquitination and degradation, these observations led us to hypothesize that OTUB1 promotes FOXM1 expression at the posttranslational level to potentiate genotoxic agent resistance.

reach
FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression.
OTUB1-C91S increases the amount of FOXM1. 1 / 1
| 1

reach
The FOXM1 co-immunoprecipitation analysis showed that overexpression of OTUB1, but not the OTUB1 (C91S) mutant, substantially depleted the levels of K48-polyubiquitin chains and induced FOXM1 expression, particularly following epirubicin treatment (XREF_FIG).
Modified OTUB1 increases the amount of FOXM1. 1 / 1
| 1

reach
The FOXM1 co-immunoprecipitation analysis showed that overexpression of OTUB1, but not the OTUB1 (C91S) mutant, substantially depleted the levels of K48-polyubiquitin chains and induced FOXM1 expression, particularly following epirubicin treatment (XREF_FIG).
OTUB1 decreases the amount of FOXM1.
| 1
OTUB1 decreases the amount of FOXM1. 1 / 3
| 1

reach
Nevertheless, the correlations between the kinetics of OTUB1 and FOXM1 expression in the sensitive and resistant MCF-7 cells in response to DNA damaging agents support the notion that OTUB1 restricts the downregulation of FOXM1 expression in response to DNA damage and genotoxic agents.
E2 affects OTUB1
| 25
E2 activates OTUB1.
| 20
E2 activates OTUB1. 10 / 20
| 20

reach
Thus, the same E2 enzymes that are inhibited by OTUB1 when the E2 is charged with ubiquitin can stimulate OTUB1 when uncharged.

reach
The effect of E2 enzymes in lowering the K M of OTUB1 for substrate suggests that the binding of an E2 partner increases the affinity of OTUB1 for K48 diubiquitin.

reach
For the DUB, we used human OTUB1, which selectively cleaves K48 Ub-Ub linkages 33 and is activated allosterically by certain E2 enzymes 34.

reach
Since the N-terminal residues of OTUB1 that precede the OTU domain form a proximal ubiquitin binding helix that plays an essential role in E2 inhibition, we asked whether E2 stimulation of OTUB1 activity similarly requires the OTUB1 N-terminus.

reach
We have shown here that a subset of E2 enzymes markedly stimulate OTUB1 cleavage of Lys48 linked polyubiquitin (XREF_FIG) and that the same set of OTUB1-E2 interactions are required for both OTUB1 non catalytic inhibition of E2 enzymes and E2 stimulation of OTUB1 XREF_BIBR, XREF_BIBR.

reach
No difference in stimulation was observed in cleavage of di-, tri- and tetraubiquitin substrates (XREF_SUPPLEMENTARY), indicating that E2 stimulation of OTUB1 DUB activity is independent of polyubiquitin chain length.

reach
Only the uncharged E2 stimulates OTUB1 DUB activity at physiological concentrations of ubiquitin 37, whereas the charged E2 ~ Ub drives formation of a repressed complex with OTUB1 XREF_BIBR, XREF_BIBR, XREF_BIBR.

reach
Importantly, the fact that a substantial proportion of the E2 partners of OTUB1 are uncharged is consistent with a role for E2 stimulation of OTUB1 in the cell.

reach
The E2 increased the affinity of OTUB1 for K48 diubiquitin, as reflected in a decrease in K d from 84 muM with no E2 to 12 muM in the presence of UBE2D1, 13.2 muM in the presence of UBE2D3, and 22.3 muM in the presence of UBE2N.

reach
Of the E2 enzymes that stimulate OTUB1 (XREF_FIG), UBCH5C (UBE2E3) and UBC13 (UBE2N) have been shown to be the most abundant and in molar excess over OTUB1 20.
E2 inhibits OTUB1.
| 5
E2 inhibits OTUB1. 5 / 5
| 5

reach
The ternary E2 ~ Ub and OTUB1/Ub complex inhibits both ubiquitin discharge from the E2 ~ Ub intermediate and the DUB activity of OTUB1 [XREF_BIBR, XREF_BIBR].

reach
Since only uncharged E2 stimulates OTUB1 DUB activity while charged E2 ~ Ub represses OTUB1 under normal cellular concentrations of free ubiquitin, the relative proportion of charged versus uncharged E2s must be critical determinants of whether OTUB1-E2 complexes function as active DUBs in the cell.

reach
Since estimates of cellular free ubiquitin concentrations range from 4 - 20 microM, this would suggest that E2 ~ Ub conjugates would repress OTUB1 activity unless cellular stresses significantly reduced the availability of free ubiquitin.

reach
The E2 mediated conformational change decreases the K m of OTUB1 for diUb by over 35-fold, thereby enhancing the rate of OTUB1 dependent polyUb degradation 2.

reach
As was previously shown for UBE2D2 37, all E2 partners tested lowered the K M of OTUB1 for its substrate (XREF_FIG), with no effect on k cat.

reach
A growing body of literature demonstrates that the DUB Otubain 1 (OTUB1) regulates many cancer associated signaling pathways including MAPK, ERa, epithelial-mesenchymal transition (EMT), RHOa, mTORC1, FOXM1 and P53 to promote tumor cell survival, proliferation, invasiveness and therapeutic resistance.

reach
We finally determined that OTUB1 promotes the proliferation and progression of PCa via deubiquitinating and stabling Cyclin E1.

reach
Activation of mTOR allows selective mRNA translation, including the epistatic regulator of GRAIL, Otubain-1 (Otub1), whose expression results in the degradation of GRAIL and allows T cell proliferation.

reach
18 In here, we not only show that the potent oncogene FOXM1 is a target of OTUB1 but also present evidence to demonstrate that the oncogenic and genotoxic functions of OTUB1 depend on the expression of a functional FOXM1, suggesting that OTUB1 promotes cell proliferation and epirubicin resistance predominantly through targeting FOXM1.

reach
The result showed that overexpression of OTUB1 significantly enhanced MCF-7 cell proliferation, whereas OTUB1 depletion decreased the rates of proliferation of MCF-7 cells (XREF_SUPPLEMENTARY).

reach
Furthermore, addition of IL-2 was able to activate the mTOR pathway, inducing Otubain-1 expression, which mediated GRAIL degradation and improved T cell proliferation.

reach
OTUB1 promotes cell proliferation and epirubicin resistance through targeting FOXM1.

reach
Conclusions : Our findings reveal the critical role of OTUB1 in PCa, and OTUB1 promotes the proliferation and progression of PCa via deubiquitinating and stabilizing Cyclin E1.

reach
Overexpression of OTUB1, but not the OTUB1 (C91S) mutant, significantly enhanced the cell proliferation as well as the viability of WT MEFs in response to epirubicin (XREF_FIG).

reach
Through in vitro and in vivo experiments, knockdown of OTUB1 suppressed PTC cells growth whereas OTUB1 overexpression enhanced the proliferation ability of PTC cells.

reach
The result showed that overexpression of OTUB1 significantly enhanced MCF-7 cell proliferation, whereas OTUB1 depletion decreased the rates of proliferation of MCF-7 cells (XREF_SUPPLEMENTARY).

reach
Flow cytometry-based analysis of E18.5 lung tissue revealed that Otub1 deletion increased proliferation of major lung parenchymal and mesenchymal/other non-hematopoietic cell types.

reach
Knockdown of OTUB1 inhibited cell viability and proliferation, as well as migration and invasion of RCC cells.

reach
Consequently, overexpression of Otub1 induces p53 dependent apoptosis and inhibition of cell proliferation whereas knockdown of Otub1 attenuates p53 activation following DNA damage [XREF_BIBR].
| 1 14
| 11

reach
As described in our previous work, Otub1 stabilizes and activates p53 and can induce apoptosis [XREF_BIBR].

reach
In addition, OTUB1 also promotes UV induced apoptosis.

reach
Otub1 promotes UV induced apoptosis that involves MDMX stabilization.

reach
Consequently, overexpression of Otub1 induces p53 dependent apoptosis and inhibition of cell proliferation whereas knockdown of Otub1 attenuates p53 activation following DNA damage [XREF_BIBR].

reach
Together, these results suggest that Otub1 stabilizes MDMX and promotes p53S46P and mitochondria mediated apoptosis, providing an alternative mechanism of Otub1 's role in apoptosis.

reach
Also, Otub1 promotes UV-irradiation-induced apoptosis, which can be inhibited by MDMX depletion.

reach
Taken together, our findings show that OTUB1 induces apoptosis of melanoma cells in vitro, likely by upregulating TRAIL, and suggest that approaches targeting OTUB1 can be developed to provide novel therapeutic strategies for treating melanoma.

reach
Together, these results suggest that Otub1 also induces mitochondrial mediated apoptosis by stabilizing MDMX and relocalizing it to mitochondria to induce mitochondria mediated apoptosis.

reach
The result showed that AVT drastically reduced the protein level of pro-caspase-3 in both RPMI-8226 and LP1 cells, but it was abolished partly by lentiviral Otub1, suggesting Otub1 expression rescued the apoptosis induced by AVT.

reach
Furthermore, Otub1 promotes UV-irradiation-induced p53S46P and apoptosis, which can be significantly inhibited by MDMX depletion.
| 1 3

reach
Inhibition of the Otub1 and c-Maf axis by the herbal acevaltrate induces myeloma cell apoptosis.

reach
OTUB1 attenuates neuronal apoptosis after intracerebral hemorrhage.

reach
Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1 LPC-KO) and human OTUB1 deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal and TNF challenge.

eidos
Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells ( OTUB1 LPC-KO ) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal / TNF challenge .
| 16

reach
While overexpression of WT OTUB1 leads to increased cell invasion, the expression of the OTUB1-C91S variant mimics the effects of OTUB1 siRNA knockdown, leading to reduced cell invasion.

reach
OTUB1 promotes cancer cell invasion and tumorigenesis in various human cancers XREF_BIBR - XREF_BIBR, while the role of OTUB2 in cancer progression remains unknown.

reach
Erratum : OTUB1 de-ubiquitinating enzyme promotes prostate cancer cell invasion in vitro and tumorigenesis in vivo.

reach
OTUB1 promotes tumor invasion and predicts a poor prognosis in gastric adenocarcinoma.

reach
OTUB1 promotes the migration and invasion of CRC cell lines.

reach
In vitro wound healing and transwell assays showed that OTUB1 overexpression promoted tumor cell migration and invasion in gastric cancer cells.

reach
Together, our findings demonstrate that OTUB1 promotes the migration, invasion, and metastasis of CRC cells in vitro and in vivo and acts as a potential metastasis marker and prognostic factor in CRC.

reach
XREF_BIBR, XREF_BIBR In addition, OTUB1 promotes tumourigenesis and cell invasion in prostate cancer.

reach
Finally, cell migration and invasion inhibition induced by miR-542-3p overexpression was partially attenuated by co-transfection of recombinant OTUB1 in KYSE150 cells.

reach
OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo.

reach
Knockdown of OTUB1 inhibited cell viability and proliferation, as well as migration and invasion of RCC cells.
OTUB1 affects MAPT
2 | 14
OTUB1 activates MAPT.
| 12
OTUB1 activates MAPT. 10 / 12
| 12

reach
Consequently, Otub1 dependent deubiquitination inhibits Tau degradation through the proteasome pathway and prolongs its half-life, leading to accumulation of pathological forms of Tau.

reach
Otub1 enhances Tau oligomerization and Tau seeded Tau aggregation in primary neurons.

reach
Otub1 increases Tau stability and increases Tau aggregation in a well characterized cellular Tau aggregation model.

reach
Taken together, our data demonstrate that Otub1, identified as a Tau interacting protein in the Tau interactome mapping, modulates Tau, by increasing Tau seeded Tau aggregation, by formation of a Otub1 induced Tau complex Tau c, and by increasing the concentration of oligomeric Tau forms in primary neurons.

reach
Knockdown of Otub1 increased Tau turnover, correlating with the decrease in expression of Otub1 obtained by the different siRNAs.

reach
Otub1 but not Otub1-C91A impairs Tau degradation by removing Lys48 linked polyubiquitin chains from Tau in primary neurons.

reach
Finally, we demonstrated that expression of Otub1 but not its catalytically inactive form induced pathological Tau forms after 2months in Tau transgenic mice invivo, including AT8 positive Tau and oligomeric Tau forms.

reach
In addition, we found robust induction of a (homo- or heterotypic) Tau complex (Tau c), which was strongly detected in AAV-Otub1-infected neurons but absent in AAV-GFP-infected neurons, indicating clearcut Otub1 induced modulation of Tau in neurons.

reach
Taken together, we conclude that overexpressed Otub1 in primary neurons expressing TauP301S increased total Tau and induced a robust increase in AT8 positive Tau.

reach
We demonstrate that Otub1 regulates levels of Lys48 linked ubiquitin conjugated Tau and increases pathological forms of Tau invitro and invivo.
OTUB1 increases the amount of MAPT.
2 | 1
OTUB1 increases the amount of MAPT. 3 / 3
2 | 1

bel
We found that Tau degradation was significantly impaired in primary neurons infected with Otub1 WT, but not with catalytically dead mutant C91A, compared with GFP control.

reach
Otub1 also significantly increased total Tau protein level compared with GFP control.

bel
Otub1 also significantly increased total Tau protein level compared with GFP control (Fig. 3b).
OTUB1 inhibits MAPT.
| 1
OTUB1 inhibits MAPT. 1 / 1
| 1

reach
Endogenous Otub1 depletion significantly inhibited Tau aggregation, with the level of inhibition correlating with the level of knockdown efficiency, while use of a control siRNA did not affect levels of Otub1 nor aggregation efficiency.
OTUB1 affects SLC7A11
| 15
OTUB1 activates SLC7A11.
| 5
OTUB1 activates SLC7A11. 5 / 5
| 5

reach
Taken together, although the precise mechanism by which OTUB1 induces SLC7A11 stabilization requires further elucidation, it is very likely that the binding between OTUB1 and SLC7A11 as well as OTUB1 's ability of inhibiting E2 conjugating enzymes recruited by the unknown E3 ligase contribute to SLC7A11 stabilization induced by OTUB1.

reach
Consistent with the role of a DUB in regulating protein stability, OTUB1 overexpression resulted in reduced SLC7A11 ubiquitination and increased SLC7A11 protein half-life and steady protein levels; conversely, OTUB1 deletion in a variety of cancer cell lines resulted in a significant decrease in SLC7A11 protein levels.

reach
Evidence suggests that the deubiquitinase OTUB1, usually overexpressed in cancers, replicates the ferroptosis process and promotes tumor development by stabilizing the cystine transporter SLC7A11 (Gan, 2019).

reach
For example, in a non neuronal setting, induction of ferroptosis with the small molecule erastin is suppressed by the ubiquitin ligase NEDD4 [XREF_BIBR], whilst the DUBs OTUB1 and USP7 promote ferroptosis by stabilising the mediators SLC7A11 and p53, respectively [XREF_BIBR, XREF_BIBR].

reach
As shown in XREF_FIG, the stability of SLC7A11 was effectively rescued by OTUB1 (C91A) but not by OTUB1 (D88A), suggesting that OTUB1 promotes SLC7A11 stabilization independent of its deubiquitinase activity.
OTUB1 increases the amount of SLC7A11.
| 5
OTUB1 increases the amount of SLC7A11. 4 / 4
| 4

reach
In particular, our results show that (i) OTUB1 is a bona fide binding partner of SLC7A11 both in vitro and in vivo; (ii) OTUB1 acts as a major regulator for SLC7A11 activity in human cancer cells; (iii) OTUB1 inactivation promotes ferroptosis in human cancer cells primarily by down-regulating SLC7A11 levels; (iv) OTUB1 is overexpressed in human cancers and the OTUB1-SLC7A11 interaction is critical for tumor growth; (v) The OTUB1-SLC7A11 interaction is tightly regulated by CD44 in human cancer cells.

reach
OTUB1 inactivation promotes ferroptosis in human cancer cells primarily by down-regulating SLC7A11 levels.

reach
Together, these data suggest that OTUB1 inactivation promotes ferroptosis in human cancer cells primarily by down-regulating SLC7A11 levels.

reach
OTUB1 directly interacted with and stabilized SLC7A11; conversely, OTUB1 knockdown diminished SLC7A11 levels in cancer cells.
Modified OTUB1 increases the amount of SLC7A11. 1 / 1
| 1

reach
As expected in T24 cells, loss of OTUB1 expression diminished the levels of endogenous SLC7A11 (XREF_SUPPLEMENTARY); ROS mediated ferroptosis was significantly enhanced in all clones of OTUB1-null cells (XREF_SUPPLEMENTARY); the same results were observed in OTUB1-null UM-UC-3 cells (XREF_SUPPLEMENTARY, XREF_SUPPLEMENTARY and XREF_SUPPLEMENTARY).
OTUB1 inhibits SLC7A11.
| 4
| 3

reach
Like CD44, OTUB1 is also overexpressed in human cancers and acts to suppress ferroptosis by promoting SLC7A11 stability.

reach
As shown in XREF_FIG, the steady-state levels of SLC7A11 in OTUB1 depleted cells were significantly restored upon treatment with the MG132 proteasome inhibitor (lane 4 vs. lane 2), indicating that OTUB1 depletion promotes SLC7A11 degradation in a proteasome dependent manner.

reach
Moreover, depletion of endogenous OTUB1 diminishes SLC7A11 activity in human cancer cells.
Modified OTUB1 inhibits SLC7A11. 1 / 1
| 1

reach
The steady-state levels of ubiquitinated SLC7A11 were also reduced by OTUB1 expression (XREF_FIG, lane 3 vs. lane 4), suggesting that OTUB1 stabilizes SLC7A11 by directly reducing its ubiquitination levels.
OTUB1 decreases the amount of SLC7A11.
| 1
Modified OTUB1 decreases the amount of ubiquitinated SLC7A11. 1 / 1
| 1

reach
The steady-state levels of ubiquitinated SLC7A11 were also reduced by OTUB1 expression (XREF_FIG, lane 3 vs. lane 4), suggesting that OTUB1 stabilizes SLC7A11 by directly reducing its ubiquitination levels.
| 11

reach
OTUB1 contributes to drug resistance and promotes metastasis.

reach
Indeed, OTUB1 has been reported to promote metastasis by facilitating EMT [XREF_BIBR].

reach
There may be a variety of mechanisms involved in OTUB1 promoting the metastasis of colorectal cancer.

reach
OTUB1 promotes metastasis and serves as a marker of poor prognosis in colorectal cancer.

reach
Together, our findings demonstrate that OTUB1 promotes the migration, invasion, and metastasis of CRC cells in vitro and in vivo and acts as a potential metastasis marker and prognostic factor in CRC.

reach
In this sense, OTUB1 may promote colon cancer proliferation, metastasis and recurrence through TGFbeta signal pathway.

reach
OTUB1 promotes CRC metastasis by facilitating EMT and acts as a potential distant metastasis marker and prognostic factor in CRC.

reach
Recently, OTUB1 was demonstrated to promote the metastasis of esophageal cancer by stabilizing Snail.

reach
OTUB1 promotes esophageal squamous cell carcinoma metastasis through modulating Snail stability.

reach
These results revealed that OTUB1 promoted CRC cell liver metastasis in vivo.
OTUB1 affects MAPK
| 10
OTUB1 activates MAPK. 10 / 13
| 10

reach
Hyperactivation of the MAPK signaling by OTUB1 overexpression suggests that OTUB1 overexpression may promote tumorigenic transformation.

reach
First, OTUB1 promotes hyperactivation of the MAPK cascade only when overexpressed in a wt RAS background.

reach
We also did not observe OTUB1 induced hyperactivation of the MAPK kinase pathway when we overexpressed a dominant negative KRAS S17N-mutant, indicating that the effect of OTUB1 overexpression is RAS dependent (XREF_SUPPLEMENTARY).

reach
Furthermore, knockdown of OTUB1 reduces TWEAK induced activation of canonical NF-kappaB and MAPK signalling pathways and modulates TWEAK induced gene expression.

reach
Here we found that OTUB1 promotes tumorigenic transformation of wt RAS cells by triggering the RAS and MAPK pathway.

reach
OTUB1 overexpression in the H1993 cell line harboring wt KRAS led to a higher and more sustained activation of ERK1/2 phosphorylation (Fig XREF_FIG A), whereas the introduction of OTUB1 into KRAS-mutant A549 cells only slightly increased the activity of the MAPK pathway (Fig XREF_FIG B).

reach
Consistently with our observation that OTUB1 overexpression induces the MAPK cascade activation in cells with wt RAS, we found that higher levels of OTUB1 significantly correlated with increased levels of ERK1/2 phosphorylation in lung adenocarcinomas harboring wt KRAS (Pearson 's coefficient : 0.352; P-value : 0.013) (Fig XREF_FIG D and E), while mutant KRAS tumors exhibited in general higher levels of ERK1/2 phosphorylation (Fig XREF_FIG D and E).

reach
On the other hand, OTUB1 did not further accelerate anchorage independent colony formation of HEK TE cells overexpressing both a constitutively active MEK1 D218, D222 allele (MEKDD) and myr-AKT, further confirming that OTUB1 overexpression promotes tumorigenic transformation by inducing the MAPK cascade activation.

reach
We observed a similar overactivation of the MAPK pathway, when we overexpressed catalytically inactive OTUB1 C91S-mutant, indicating that catalytic activity of OTUB1 is not necessary to induce the MAPK pathway activation (Fig XREF_FIG F).

reach
Given that OTUB1 overexpression up-regulated the MAPK pathway, but did not affect AKT signaling, we hypothesized that OTUB1 could cooperate with myristoylated (myr) and therefore the constitutively active allele of AKT1 (myr-AKT) to promote cell transformation.
| 11

eidos
OTUB1 Promotes Progression and Proliferation of Prostate Cancer via Deubiquitinating and Stabling Cyclin E1 Background : Prostate cancer ( PCa ) is currently the most common cancer among males worldwide .

eidos
So far , we have a lot of evidence to identify the hypothesis that OTUB1 promotes the progression and proliferation of prostate cancer via mediating and stabling Cyclin E1 .

eidos
These results suggested that OTUB1 might promote the proliferation of PCa via mediating Cyclin E1 .

eidos
Conclusions : Our findings reveal the critical role of OTUB1 in PCa , and OTUB1 promotes the proliferation and progression of PCa via deubiquitinating and stabilizing Cyclin E1 .

eidos
OTUB1 promotes prostate cancer [ 12 ] .

eidos
And we further found that OTUB1 promoted the progression of PCa via regulating the stability and interacting with Cyclin E1 .

eidos
Discussion In our research , we found that the expression levels of OTUB1 are up-regulated in PCa , OTUB1 could promote the proliferation and progression of PCa via deubiquitinating and stabling the expression of Cyclin E1 protein .

eidos
We finally determined that OTUB1 promotes the proliferation and progression of PCa via deubiquitinating and stabling Cyclin E1 .

eidos
Therefore , this research preliminarily proposed that OTUB1 could promote the progression and proliferation of PCa via regulating the expression of Cyclin E1 , thus the specific internal mechanism will become the main work and direction in the next step .

eidos
OTUB1 Promotes Progression and Proliferation of Prostate Cancer via Deubiquitinating and Stabling Cyclin E1 .
OTUB1 affects TGFB
| 9
OTUB1 activates TGFB.
| 6
OTUB1 activates TGFB. 5 / 7
| 5

reach
However, different from USP15, OTUB1 enhances TGF-beta signaling by inhibiting the ubiquitination and degradation of active SMAD2/3 (and not the inactive un phosphorylated form), because the association of OTUB1 to SMAD2/3 is phosphorylation dependent.

reach
OTUB1 has been found to modulate TGFbeta signalling by promoting the stabilization of only the phosphorylated form of SMAD2/3 [98].

reach
Our findings indicate that OTUB1 modulates TGFbeta induced transcriptional responses.

reach
These results suggest that Otub1 enhances TGFbeta induced transcriptional responses.

reach
OTUB1 activates TGF-beta signaling via activating (phospho-) SMAD2/3.
Ubiquitinated OTUB1 activates TGFB. 1 / 1
| 1

reach
In contrast, OTU domain, ubiquitin aldehyde binding 1 (OTUB1), B-cell lymphoma-3 (BCL-3), ubiquitin carboxyl-terminal hydrolase 1 (UCHL1), and UCHL5 contribute to the deubiquitination of SMAD2 or SMAD3, making them more stable and less easily degradable and promoting TGF-beta signaling [XREF_BIBR - XREF_BIBR].
| PMC
OTUB1 increases the amount of TGFB.
| 3
OTUB1 increases the amount of TGFB. 3 / 3
| 3

reach
OTUB1 enhances TGFbeta mediated gene transcription.

reach
However, we must acknowledge that we did not make a long term follow-up and survival analysis, since most patients accepted varieties of postoperative chemotherapy schemes, including traditional Chine[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

reach
We tested whether the catalytic activity of OTUB1, its ability to inhibit ubiquitylation of SMAD2/3 or its ability to interact with SMAD3 are essential to enhance TGFbeta induced transcription.
OTUB1 affects UBE2
| 3
OTUB1 inhibits UBE2. 3 / 10
| 3

sparser
Thus, the same E2 enzymes that are inhibited by OTUB1 when the E2 is charged with ubiquitin can stimulate OTUB1 when uncharged.

sparser
In addition, OTUB1 inhibits E2 ubiquitin-conjugating enzymes independent of its enzymatic activity, impeding ubiquitin chain formation [ xref , xref , xref ].

sparser
We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners.
OTUB1 affects SMAD2_3
| 2
OTUB1 inhibits SMAD2_3.
| 1
| 1

reach
Therefore, the association of OTUB1 with phospho-SMAD2/3 would be predicted to prevent SMAD2/3 from being polyubiquitylated and degraded, thereby enhancing TGFbeta signalling.
OTUB1 activates SMAD2_3.
| 1
OTUB1 activates SMAD2_3. 1 / 4
| 1

reach
OTUB1 has been found to modulate TGFbeta signalling by promoting the stabilization of only the phosphorylated form of SMAD2/3 [98].
OTUB1 affects UBE2D2
| 2 5
OTUB1 inhibits UBE2D2. 7 / 9
| 2 5

reach
Interestingly, this non canonical mode of action by OTUB1 was found not to be unique to UBC13, as OTUB1 was also found to interact with and inhibit the E2 enzymes UBE2D2 and UBCH5B and UBE2D3 and UBCH[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]

reach
Competition with E3 binding is likely to be particularly important for OTUB1 inhibition of UBCH5b, which, unlike UBC13, is strictly dependent upon an E3 ligase for activity.

reach
Since OTUB1 also inhibits UBCH5b 4, which does not function with a UEV, we speculate that the OTUB1 N-terminus may also interfere with acceptor ubiquitin binding for other E2s.

sparser
Competition with E3 binding is likely to be particularly important for OTUB1 inhibition of UBCH5b, which, unlike UBC13, is strictly dependent upon an E3 ligase for activity.

reach
The OTUB1 double E28A D35A substitution, which affects OTUB1 contacts with UBCH5B, greatly decreased the ability of UBCH5B to stimulate OTUB1 DUB activity without affecting OTUB1 activity in the absence of UBCH5B (XREF_FIG).

sparser
Similar interactions could form between OTUB1 and UBE2D2 (UBCH5b) ( xref ), but clashes due to an insertion and a non-conserved lysine would arise with UBE2L3 (UBCH7), consistent with the observation that OTUB1 inhibits UBCH5b but not UBCH7 xref .

reach
Similar interactions could form between OTUB1 and UBE2D2 (UBCH5b) (XREF_FIG), but clashes due to an insertion and a non conserved lysine would arise with UBE2L3 (UBCH7), consistent with the observation that OTUB1 inhibits UBCH5b but not UBCH7 4.
OTUB1 affects UBE2D1
| 1 3 3
OTUB1 inhibits UBE2D1. 7 / 9
| 1 3 3

sparser
One such example is OTUB1 which can inhibit UbcH5.

sparser
In this study, we sought to elucidate the structural mechanism by which OTUB1 recognizes and inhibits UBC13, UbcH5 and related E2 enzymes.

trips
Otub1 regulates p53 stability and activity via non-canonical inhibition of UbcH5, the MDM2 cognate ubiquitin-conjugating enzyme (E2).

reach
17 OTUB1 also suppresses UBCH5 E2 enzyme and stabilizes the p53 protein.

sparser
It has been reported that OTUB1 non-catalytically inhibits UBE2D proteins from ubiquitinating p53 xref , xref and SMAD2/3, regulators of TGFβ signaling xref .

reach
One such example is OTUB1 which can inhibit UbcH5.

reach
Monoubiquitination is critical for ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (Otub1) to suppress UbcH5 enzyme and stabilize p53 protein.
UBE2D2 affects OTUB1
| 6
UBE2D2 activates OTUB1.
| 5
UBE2D2 activates OTUB1. 5 / 8
| 5

reach
To confirm that the observed stimulation was not assay dependent, we used a gel based assay to verify that UBCH5B stimulates OTUB1 cleavage of native Lys48 linked diubiquitin (XREF_FIG and XREF_SUPPLEMENTARY).

reach
Thes results indicate that UBCH5B stimulates OTUB1 by increasing its affinity for the Lys48 diubiquitin substrate.

reach
We assayed the ability of UBCH5B ~ Ub to stimulate OTUB1 (XREF_FIG) and found that both charged and uncharged UBCH5B stimulate OTUB1 to a similar degree.

reach
The importance of the OTUB1 N-terminal helix to its ability to be stimulated by UBCH5B, together with the role of these N-terminal residues in forming the proximal ubiquitin binding site of OTUB1 XREF_BIBR, XREF_BIBR, XREF_BIBR, suggested that UBCH5B may increase OTUB1 affinity for Lys48 diubiquitin via the proximal ubiquitin binding site.

reach
However, assays of OTUB1 stimulation by UBCH5B or UBCH5B ~ Ub over a range of ubiquitin concentrations (XREF_FIG) (XREF_SUPPLEMENTARY) showed that stimulation by UBCH5B is relatively insensitive to free ubiquitin, but that stimulation by UBCH5B ~ Ub decreases as a function of increasing concentration of free ubiquitin, dropping to low levels below ~ 1 microM free ubiquitin.
UBE2D2 inhibits OTUB1.
| 1
UBE2D2 inhibits OTUB1. 1 / 1
| 1

reach
A previous study 37 reported that the binding of UBE2D2 lowered the K M of OTUB1 for K48 diubiquitin by 34-fold, from 120 muM to 3.4 muM, with no significant change in k cat.
OTUB1 affects RNF128
| 9
OTUB1 inhibits RNF128.
| 5
OTUB1 inhibits RNF128. 5 / 5
| 5

reach
Furthermore, addition of IL-2 was able to activate the mTOR pathway, inducing Otubain-1 expression, which mediated GRAIL degradation and improved T cell proliferation.

reach
Thus, our current working model is that Otub 1 promotes GRAIL degradation by de-ubiquitination of ubiquitinated USP8, thereby diminishing USP8 activity (XREF_FIG).

reach
Interestingly, Otub1 expression completely abolished USP8 mediated stabilization of GRAIL when all 3 proteins were co-expressed.

reach
Whereas otubain-1 negatively regulates GRAIL in CD4 + T cells, otubain-1 ARF-1 stabilizes GRAIL expression.

reach
Otub1 forms a trimolecular complex with USP8 and GRAIL, and upregulated Otub1 promotes GRAIL 's proteosomal degradation.
OTUB1 activates RNF128.
| 4
OTUB1 activates RNF128. 4 / 4
| 4

reach
As Otub-1 is controlled by the Akt-mTOR pathway and is a negative regulator of the GRAIL function [XREF_BIBR, XREF_BIBR], this suggests that T. cruzi infection may disrupt the Akt-mTOR pathway resulting in Otub-1 downregulation, which in turn may induce GRAIL.

reach
Moreover, the catalytic inactive C748S USP8 mutant made no difference on Otub1 mediated GRAIL stability.

reach
This suggests that LAM may disrupt the Akt-mTOR pathway resulting in Otub-1 down-regulation, which in turn may induce GRAIL.

reach
When mTOR is inhibited by rapamycin treatment during stimulation, although Otub1 mRNA is up-regulated, Otub1 protein is not expressed, allowing GRAIL to be maintained (XREF_FIG, third lane).
OTUB1 affects NFkappaB
| 1 6
OTUB1 activates NFkappaB.
| 1 4
| 1 4

reach
Mechanistically, OTUB1 promoted NF-kappaB activity in DCs by K48 linked deubiquitination and stabilization of the E2 conjugating enzyme UBC13, resulting in increased K63 linked ubiquitination of IRAK1 (IL-1 receptor associated kinase 1) and TRAF6 (TNF receptor associated factor 6).

reach
Otub1 deficiency not only promotes signal induced p100 processing and noncanonical NF-kappaB activation but also causes steady-state p100 degradation, leading to aberrant NF-kappaB activation in the canonical pathway.

reach
Furthermore, knockdown of OTUB1 reduces TWEAK induced activation of canonical NF-kappaB and MAPK signalling pathways and modulates TWEAK induced gene expression.

reach
Recently, it was also observed that OTUB-1 promotes NF-kappaB activation in dendritic cells during stimulated inflammatory conditions by lysine48 deubiquitination of UBC13 [XREF_BIBR].

sparser
Recent reports showed that TAK1 kinase and OTUB1 controls NF-κB activation using similar Ube2v1-UBC13/TRAF6 signaling axis.
OTUB1 inhibits NFkappaB.
| 2
| 2

reach
Overexpression of OTUB1 and OTUB2 inhibited virus induced activation of IRF3 and NF-kappaB, transcription of the IFNB1 gene as well as cellular antiviral response, whereas knockdown of OTUB1 and OTUB2 had opposite effects.

reach
The overexpression of OTUB1 inhibited virus induced activation of IRF3 and NF-kappaB, whereas knockdown of OTUB1 had the opposite effects.
OTUB1 affects MDM4
| 9
OTUB1 increases the amount of MDM4.
| 6
OTUB1 increases the amount of MDM4. 5 / 5
| 5

reach
These results suggest that Otub1 increases the cytoplasmic levels of both MDMX and p53.

reach
Overexpression of Otub1 markedly stabilized MDMX and induced its levels, whereas knockdown of Otub1 reduced the levels of MDMX.

reach
Consistently, knockdown of endogenous Otub1 by two different siRNAs significantly reduced the levels of MDMX in cells.

reach
Consequently, overexpression of Otub1 markedly stabilized MDMX and increased its levels, whereas knockdown of Otub1 reduced the levels of MDMX.

reach
Doxycycline induced expression of Otub1 in T-Rex-U2OS-Flag-Otub1 cells also drastically induced the levels of MDMX, as well as p53 and its target MDM2 in a time dependent manner.
Modified OTUB1 increases the amount of MDM4. 1 / 1
| 1

reach
As shown in Figure XREF_FIG, cell fractionation assays revealed that Otub1 expression not only significantly induced the nuclear levels of p53 and MDMX, but also induced the levels of cytoplasmic MDMX and p53 (lane 4, Figure XREF_FIG).
OTUB1 activates MDM4.
| 3
OTUB1 activates MDM4. 3 / 3
| 3

reach
Interestingly, MDMX induced by Otub1 can localize to mitochondria in addition to the cytosol, enhance p53 phosphorylation at S46 (p53S46P) and promote mitochondria mediated apoptotic pathway.

reach
Also, induced overexpression of Otub1 drastically prolonged the half-life of MDMX as determined by half-life assays.

reach
Together, these results suggest that Otub1 also induces mitochondrial mediated apoptosis by stabilizing MDMX and relocalizing it to mitochondria to induce mitochondria mediated apoptosis.
| 9
| 8

reach
Overexpression of OTUB1, but not the OTUB1 (C91S) mutant, significantly enhanced the cell proliferation as well as the viability of WT MEFs in response to epirubicin (XREF_FIG).

reach
Furthermore, we also found that OTUB1 overexpression enhanced the resistance of MCF-7 cells to epirubicin (XREF_SUPPLEMENTARY).

reach
Our data show that WT OTUB1, but not the OTUB1 (C91S) catalytic dead mutant, can enhance the stability of FOXM1 in response to epirubicin in MCF-7 cells, suggesting that the deubiquitinase activity is required for the degradation of FOXM1.

reach
OTUB1 promotes cell proliferation and epirubicin resistance through targeting FOXM1.

reach
18 In here, we not only show that the potent oncogene FOXM1 is a target of OTUB1 but also present evidence to demonstrate that the oncogenic and genotoxic functions of OTUB1 depend on the expression of a functional FOXM1, suggesting that OTUB1 promotes cell proliferation and epirubicin resistance predominantly through targeting FOXM1.

reach
In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1 deficient cells.
Modified OTUB1 activates 4'-epidoxorubicin. 1 / 1
| 1

reach
The FOXM1 co-immunoprecipitation analysis showed that overexpression of OTUB1, but not the OTUB1 (C91S) mutant, substantially depleted the levels of K48-polyubiquitin chains and induced FOXM1 expression, particularly following epirubicin treatment (XREF_FIG).
| 1

reach
Our data show that WT OTUB1, but not the OTUB1 (C91S) catalytic dead mutant, can enhance the stability of FOXM1 in response to epirubicin in MCF-7 cells, suggesting that the deubiquitinase activity is required for the degradation of FOXM1.
| 1

reach
Conversely, OTUB1 silencing potentiated the anti-proliferative function of epirubicin in MCF-7 cells (XREF_SUPPLEMENTARY).
Vasotocin affects OTUB1
| 2 5
Vasotocin inhibits OTUB1.
| 2 3
| 2 3

eidos
All cell lysates were subjected to WB assays as indicated AVT interferes with the interaction between Otub1 and c-Maf to increase c-Maf polyubiquitination AVT was identified from the Otub1 / c-Maf system , while Otub1 interacts with c-Maf and prevents its polyubiquitination as its deubiquitinase , therefore , we wondered whether AVT can inhibit Otub1 activity toward c-Maf ubiquitination .

eidos
As shown in Fig. 5b , Otub1 markedly reduced c-Maf polyubiquitination level but it was reversed by AVT , suggesting AVT inhibited Otub1 activity thus increasing the polyubiquitination level of c-Maf .

reach
AVT was identified from the Otub1 and c-Maf system, while Otub1 interacts with c-Maf and prevents its polyubiquitination as its deubiquitinase, therefore, we wondered whether AVT can inhibit Otub1 activity toward c-Maf ubiquitination.

reach
The natural product AVT inhibits the Otub1 and c-Maf axis and displays potent anti-myeloma activity.

reach
It is also in agreement with our hypothesis that AVT inhibits the Otub1 and c-Maf axis therefore leading to MM cell apoptosis and myeloma regression.
Vasotocin activates OTUB1.
| 2
| 2

reach
XREF_FIG d, AVT elicited potent activity to downregulate all these three Dubs in RPMI-8226 and LP1 but not in KMS11, suggesting that AVT might also target other Dubs in addition to Otub1, however, whether these Dubs are associated with c-Maf should be further studied.

reach
To this end, we first evaluated c-Maf ubiquitination in HEK293T cells that expressed Otub1, c-Maf and Ub, and further treated with AVT.
Ubiquitin affects OTUB1
| 4
Ubiquitin activates OTUB1.
| 1
| 1