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 USP14 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
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rlimsp
isi
tees
geneways
eidos
trips
medscan
sparser
reach
To test direct deubiquitination of Dvl by Usp14, we performed a ubiquitin chain trimming assay using immunopurified Dvl-ubiquitin conjugates and recombinant Usp14 in a proteasome-free condition (XREF_FIG and XREF_SUPPLEMENTARY).
As a lack of Dvl deubiquitination by Usp14 appears to attenuate Wnt signaling, we proposed that an elevated forward rate of ubiquitination, which will be counteracted by Usp14 activity, might be induced by Wnt3a CM treatment.
These data suggest that the USP14 gene product indeed serves as a catalyst to deubiquitinate the CXCR4, because when its expression is reduced there is a reciprocal increase in CXCR4 ubiquitination.
The physical interaction of CXCR4 and USP14 is paralleled by USP14 catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination.
In summary, our findings demonstrate that CXCL12 activation of the CXCR4 leads to a dynamic ubiquitination and deubiquitination cycle and that USP14 preferentially interacts with and deubiquitinates CXCR4.
Deubiquitination of CXCR4 by USP14 would thus be expected to reduce the rate of ligand accelerated receptor degradation and result in an increased steady state level of receptors.
In addition, reduction of UCHL5 by its siRNA did not affect the expression of AR, suggesting that USP14 but not UCHL5 recruited on the19S proteasome plays a selective role in the deubiquitination of AR.
We found that IU1 and USP14 knockdown dramatically increased levels of ubiquitinated and K48 ubiquitinated AR, suggesting that USP14 is an AR DUB, capable of deubiquitinating and thereby stabilizing AR protein.
Treatment of control MDA-MB-453 (either scramble shRNA or parental) cells with CHX for up to 12h caused decreased levels of AR, suggesting a contribution of AR protein synthesis to endogenous AR protein levels; however, co-treatment of CHX and USP14 shRNA or IU1 resulted more rapid decrease in levels of endogenous AR protein, strongly suggest that deubiquitination of AR protein by USP14 is essential for its protein stability.
Additionally, both genetic and pharmacological inhibition of USP14 significantly suppressed cell proliferation in AR-responsive breast cancer cells by blocking G0/G1 to S phase transition and inducing apoptosis.
Moreover, AR overexpression inhibited USP14 inhibition induced events, suggesting that AR deubiquitination by USP14 is critical for breast cancer growth and USP14 inhibition is a possible strategy to treat AR positive breast cancer.
In an effort to elucidate the mechanisms underlying this regulation, the Cui group showed that TLR4 stimulation activates the TRAF2/6 complex, which ubiquitinates NLRC5 on Lys1178 residue, presumably leading to its degradation and release of IKKalpha and IKKbeta to complex with IKKgamma [XREF_BIBR, XREF_BIBR] (XREF_FIG) This study also showed that the ubiquitin specific protease 14 (USP14) deubiquitinates NLRC5 to sustain the NLRC5 mediated inhibition of NF-kappaB activation.
The researchers propose that, after the ubiquitination of NLRC5 at lysine 1178 is catalyzed by TRAF2/6, USP14 specifically removes the polyubiquitin chains from NLRC5 to enhance NLRC5-mediated inhibition of IKK–NF-κB signaling, thus forming a coherent feedforward loop to regulate IKK–NF-κB activation
Taken together, our data demonstrate that USP14 can negatively regulate autophagy induction by inhibiting Beclin 1 ubiquitination, interrupting association between TRAF6 and Beclin 1, and affecting TLR4-induced activation of NF-魏B through deubiquitination of TAB 2 protein
Since USP14 induced the deubiquitination of TAB 2, we then examined the functional regulation of TLR4 mediated signaling in Ctrl and USP14 KD THP-1 cells.
Based on these previous findings, we hypothesized that the suppression of Beclin 1 ubiquitination by USP14 might be critically associated with TRAF6 mediated ubiquitination in both autophagy and TLR4 mediated signaling.
TRIM14 has been reported to interact with cGAS via its PRYSPRY domain and upon DNA virus infection recruit the proteasome-associated deubiquitinase (DUB) USP14 to deubiquitinate cGAS, preventing recruitment of p62 and autophagy-dependent degradation of cGAS (Jia et al., 2017) .
TRIM14 has been reported to interact with cGAS via its PRYSPRY domain and upon DNA virus infection recruit the proteasome associated deubiquitinase (DUB) USP14 to deubiquitinate cGAS, preventing recruitment of p62 and autophagy dependent degradation of cGAS.
Given that proteasome deubiquitination mediated by USP14 has fundamental roles in regulating proteasomal degradation of ubiquitinylated substrates XREF_BIBR XREF_BIBR, it is rational to speculate that perturbation of ubiquitin chain trimming functions of PfUSP14 may impact intraerythrocytic parasite development and cause difficulties for parasite egress from the host cell.
To investigate whether USP14 deubiquitylated CBP, we transfected MLE12 cells to overexpress USP14 or treated them with IU1, and then we examined the ubiquitylation of CBP.
Furthermore, USP14 can also deubiquitinate and stabilize vimentin, a vital protein which involves in epithelial-to-mesenchymal transition(EMT) and significantly promotes cell growth, migration and invasion in human gastric cancer
According to the above results, USP14 de-ubiquitinates vimentin and increases its expression levels, which may influence the aggressiveness of GC cells.
The physical interaction of CXCR4 and USP14 is paralleled by USP14 catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination.
Proteasomes function under tonic inhibitory conditions, possibly via the ubiquitin chain-trimming function of USP14, a proteasome-associated deubiquitinating enzyme (DUB).
Genetic and chemical suppression of USP14 activity caused an increase in Dishevelled (Dvl) polyubiquitination and significantly impaired downstream Wnt signaling, suggesting an oncogenic role for USP14 through Wnt and beta-catenin signaling enhancement [XREF_BIBR].
Consequently, USP14 deficiency may cause ataxia in the ax J mice in part by perturbing the turnover and/or cell surface distribution of GABA A R. Still, since it is not clear that USP14 directly deubiquitinates GABA A R, it is also worth considering that this DUB may regulate the receptor indirectly.
Among the potential substrates identified, we show that fatty acid synthase (FASN), a key enzyme involved in hepatic lipogenesis, is a bona fide substrate of USP14.
n this study, we demonstrated USP14, a deubiquitinating enzyme, as a negative regulator in antiviral responses by directly deubiquitinating K63-linked RIG-I.
USP14 deubiquitinated this form of NCB1 at a rate comparable to that of wild-type conjugates, indicating that USP14 does not act obligatorily on ubiquitin-ubiquitin linkages (XREF_FIG).
This study suggests that USP14 removes the ubiquitin chain of I-κB, therefore inducing I-κB degradation and increasing cytokine release in lung epithelial cells.
Our recent discovery of USP14 inhibitors clearly suggests that proteasomal DUBs can be valid pharmacological targets because USP14 inhibition may potentiate proteasome mediated protein quality control.
USP14 as a component of proteasome-associated deubiquitinating enzyme complex can eliminate ubiquitins from proteasome-bound substrates and inhibit the proteasome non-catalytically ( Chen et al ., 2018 ) .
However, USP14 does not appear to strongly antagonize proteasome function in Xenopus extract, as treatment of extract with UbVS or the USP14 specific inhibitor IU1 did not appreciably enhance turnover of pre-ubiquitinated cyclin.
Proteasome dysfunction induced by the loss of Usp14 results in a significant increase in the levels of phosphorylated tau in the brains of the ax J mice.
To verify that Usp14 inhibits the proteasome in cells, we expressed Usp14 variants in usp14 -/- murine embryonic fibroblasts (MEFs), together with proteasome substrates.
These results indicate that altered proteasome function caused by the loss of Usp14 results in widespread changes in the levels of activated stress kinases that have been implicated in tau phosphorylation.
We identified three specific RNA aptamers of USP14 (USP14-1, USP14-2, and USP14-3) that inhibited its deubiquitinating activity. The nucleotide sequences of these non-cytotoxic USP14 aptamers contained conserved GGAGG motifs, with G-rich regions upstream, and similar secondary structures. They efficiently elevated proteasomal activity, as determined by the increased degradation of small fluorogenic peptide substrates and physiological polyubiquitinated Sic1 proteins. Additionally, proteasomal degradation of tau proteins was facilitated in the presence of the UPS14 aptamers in vitro.
Loss of Ubp6 function, for example, increases aneuploidy tolerance in yeast, presumably due to an elevated proteasome capacity for turning over higher protein levels, and pharmacological inhibition of Usp14 in human cells has been shown to stimulate proteasome activity XREF_BIBR - XREF_BIBR.
These results strongly suggest that the USP14 mediated proteasome activity regulation may be directly related to various human diseases including cancer.
Recently, a small chemical compound (IU1) capable of inhibiting USP14 deubiquitination was shown to enhance proteasome mediated degradation of some substrates, including several proteins associated with neural degenerative diseases [XREF_BIBR].
Conversely, USP14 might also activate proteolysis by degrading ubiquitin chains on target proteins and thereby enhance gate opening of the 20S proteasome.
Therefore, USP14 aptamers could be used to understand molecular mechanisms of USP14 activity and Ub homeostasis in cells.How to cite this article: Lee, J. H. et al. Facilitated Tau Degradation by USP14 Aptamers via Enhanced Proteasome Activity.
In contrast, siRNA of either UCHL5 or USP14 alone did not affect proteasome composition but did increase the rate of proteasome activity, supporting previous studies.
This mutual interaction of USP14 and Ubp6 with the proteasome is thought to enhance selectivity of the proteasome for ubiquitinated proteins and couple deubiquitination to degradation.
Recently, researches have revealed USP14 enhances cisplatin resistance through affecting Akt and ERK signaling pathways and accelerates cell proliferation and migration in GC.
In addition, b-AP15, a novel inhibitor of USP14, selectively blocks the deubiquitylating activity of USP14, decreases viability and inhibits proliferation of MM cells, which is associated with growth [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
To explore the underlying mechanism by which USP14 promotes cell proliferation in LNcap cells, we monitored the cell cycle progression of each group exposed to various concentrations of IU1 (25, 50, 100muM) and found that inhibition of USP14 activity dramatically induced G0/G1 cell cycle arrest at different time points (0, 6, 12, 24, 48h) (XREF_FIG).
Overexpression of USP14 promotes cell proliferation and migration, while down-regulation induces cell apoptosis and inhibits cell proliferation, migration, and invasion.
We found that USP14 inhibition by shRNA significantly suppressed the proliferation of MDA-MB-231, MDA-MB-453, MDA-MB-468, HCC1937, and MCF7 breast cancer cell lines; however USP14 knockdown did not affect the proliferation of T47D cells, which could be related to the fact that these cells express very high levels of ER and very low levels of AR.
Recently , researches have revealed USP14 enhances cisplatin resistance through affecting Akt / ERK signaling pathways and accelerates cell proliferation and migration in GC ( Fu et al ., 2018 ; Han K.H .
USP14 was overexpressed in many cancers and promoted tumor cell proliferation through enhancing beta-catenin accumulation and inhibiting Bcl-xl-mediated cell apoptosis 11.
Conversely, overexpression of USP14 induced increases in the protein level of cyclinD1and CDK6/4/2, the inactivation of Rb, and decreases in the expression of p27 and p15 (XREF_FIG), and led to increased proliferation of LNcap cells (XREF_FIG).
These data suggest that knockdown of USP14 inhibits the proliferation and tumorigenesis in ESCC cells by suppressing and inhibiting the Wnt and beta-catenin signaling pathway.
We found that (i) USP14 could bind to AR, and additionally, both genetic and pharmacological inhibition of USP14 accelerated the ubiquitination and degradation of AR; (ii) downregulation or inhibition of USP14 suppressed cell proliferation and colony formation of LNcap cells and, conversely, overexpression of USP14 promoted the proliferation; and (iii) reduction or inhibition of USP14 induced G0/G1 phase arrest in LNcap prostate cancer cells.
Knockdown of USP14 with the lentiviral vector delivery of shRNA in human hepatocarcinoma SMMC7721 cells suppressed cell proliferation, altered the cell cycle and induced cell apoptosis.
For example, Zhu et al. reported that expression of USP14 was increased in breast cancer tissues, and downregulation of USP14 significantly inhibited breast cancer cell proliferation and metastasis XREF_BIBR.
USP14 impedes degradation of ubiquitinated proteins by removing ubiquitin chains from its substrates, while it could promote protein degradation via increasing proteasome activation.
In agreement with this data, deletion of the mouse ortholog USP14 causes a depletion of free ubiquitin and ataxic mice (ax J) that contain a mutant form of the Usp14 gene display a reduction in free monoubiquitin levels in the brain.
Hence, a more likely explanation for these genetic interactions between ubp6 Delta and the proteasome subunits is that association of Ubp6 with the mutant 26 S proteasomes stabilises these otherwise l[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
It has been reported that both USP14 and UCH37 prevent substrate degradation by removing ubiquitin chains and promoting proteasomal substrate dissociation.
By contrast, USP14 and UCHL5 are located further from the 20S core and antagonize degradation by removing Ub in a stepwise manner from the distal end, promoting substrate dissociation from the proteasome [17] .
However, later work has shown that ubiquitin overexpression does not correct the ax J deficits in hippocampal short term plasticity, and that transgenic expression of a catalytically inactive form of USP14 in the nervous system mimics the neuromuscular phenotype observed in the ax J mice, but causes a only a modest reduction of free ubiquitin.
Ubiquitin specific protease-14 (USP14), a DUB reversibly associated with the proteasome, negatively regulates the activity of proteasomes by trimming ubiquitin chains on proteasome bound substrates.
Taken together, these data show that phosphorylation of USP14 by Akt is important for this kinase to negatively regulate the UPS in a ubiquitin dependent manner.
The binding of USP14 to Rpn1 subunit through Ubl enhances its deubiquitinating activity, which is responsible for the removement of ubiquitin chains and prevent the release of ubiquitin to the proteolytic channel.
It appears that POH1 cleaves at the base of the ubiquitin chain where it is linked to the target protein, whereas USP14 and UCHL5 mediate a stepwise removal of ubiquitin from the protein by disassembling the chain from its distal tip [XREF_BIBR].
In yeast and mammals, loss of USP14 and Ubp6 results in increased degradation of ubiquitin and decreased levels of monomeric Ub, suggesting that one function of USP14 is to recycle ubiquitin at the proteasome [XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR].
Here we show that Usp14, a proteasome associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin protein conjugates, in vivo and in vitro.
These results strongly suggest that inhibition of USP14 by RNA aptamers might antagonize Ub chain-trimming on proteasomes, consequently facilitating the degradation of many UPS substrates.We investigated the effects of USP14 aptamers on HeLa cell viability.
By regulating ubiquitin pools at the nerve terminal, Usp14 would therefore be able to modulate ubiquitin dependent processes required during synaptic development.
However, as UCHL5 and USP14 are proposed to mediate a stepwise removal of ubiquitin from the substrate by trimming the chain from its distal tip, this leaves the opportunity for MGRN1 to reach a monou[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Here we show that USP14, a proteasome associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin protein conjugates both in vitro and in cells.
Similarly, inhibition of USP14 alone by IU1 significantly increased the accumulation of polyubiquitinated proteins by 22.51 +/- 4.44% (P = 0.025, n = 5) and decreased the levels of monomeric ubiquitin[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Free Ub, but not Ub conjugate, levels were reduced in the brain of ax J mice, but neuron specific expression of the Usp14 transgene was sufficient to restore free Ub levels in the spinal cord and motor neuron axons and could rescue ataxia phenotypes with defective neuromuscular junctions in ax J mice XREF_BIBR, XREF_BIBR.
Biochemical analysis showed that the ubiquitin hydrolyase activity of this form of Usp14 is dependent on the presence of proteasomes, and neuronal expression of full-length Usp14 was able to restore the levels of monomeric ubiquitin in the brains of axJ mice.
Analysis of spinal cord and sciatic nerve extracts from the ax J -Tg mice demonstrated that transgenic expression of Usp14 restored monomeric ubiquitin levels back to wt levels (XREF_FIG), indicating that Usp14 is required for the maintenance of monomeric ubiquitin in the spinal cord and motor neuron axons.
We found that USP14 inhibition significantly induced apoptosis, as evident by PARP cleavage, and downregulation of the anti-apoptotic protein Bcl-2, in AR + / ER - breast cancer, and moderately induced apoptosis in AR + / ER + MCF7 cells.
b-AP15, a small molecule inhibitor of two 19S regulatory-particle-associated deubiquitinases, USP14 and ubiquitin C-terminal hydrolase 5, could efficiently induce cell apoptosis or cell death in colorectal cancer cell line HCT116.24 In addition, b-AP15 can suppress the growth of FaDu squamous cell carcinoma cells.25 Our stratified survival analysis indicated that high USP14 expression could distinguish poor outcomes of patients with either early (TNM stage I-II) or advanced clinical stage (TNM stage III-IV), suggesting that USP14 may play a significant role throughout the development of ESCC.
Pharmacologic inhibition of USP14 and UCHL5 with VLX1570 induces apoptosis in drug resistant WM cells and is associated with accumulation of high-molecular-weight polyubiquitinated protein conjugates.
Recently, novel small molecule inhibitors of the deubiquitylating enzymes USP14 and UCHL5 were developed to overcome bortezomib resistance and induce cell apoptosis of multiple myeloma XREF_BIBR, XREF_BIBR.
Loss of USP14 expression and function dramatically decreased AR level, blocked G 0 / G 1 to S phase transition, and triggered cell apoptosis in AR + breast cancer cells, suggesting that targeting USP14/AR axis could be a potential strategy for TNBC therapy.
However, this event was markedly abolished by ATG5 knockdown, subsequently restoring the cell proliferation in IR incubated OSCC cells.Finally, we found that USP14 mediated apoptosis was autophagy dependent in IR treated OSCC cells.
Our recent study exemplifies the feasibility of such an approach : specifically, we showed that blockade of 19S associated DUBs USP14 and UCHL5 with a small-molecule inhibitor (bAP15 and VLX1570) induces apoptosis in MM cells and overcome bortezomib resistance, with a favorable toxicity profile.
Together, this confirmed that USP14 knockdown could resist OSCC cell apoptosis induced by b-AP15 treatment, which indicated that USP14 might be involved in b-AP15-induced apoptosis, further demonstrat[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The combination of enzalutamide, a nonsteroidal antiandrogen, with either knockdown or pharmacological inhibition of USP14 promotes arrest of cell cycle progression and induces apoptosis (Xia et al. 2019).
We previously confirmed that a specific USP14 and UCH37 inhibitor b-AP15 4 inhibited tumor cell growth and induced apoptosis and in vitro (data not shown).
Selective USP14 and UCHL5 inhibitor b-AP15, induced apoptosis in MM cell lines and in primary MM cells via downregulation of cell division cycle 25C (CDC25C), CDC2, and cyclin-B1, as well as the activation of caspases and unfolded protein response pathways (p-IREalpha, p-eIF2alpha, and CHOP).
Additionally, both genetic and pharmacological inhibition of USP14 significantly suppressed cell proliferation in AR responsive breast cancer cells by blocking G 0 / G 1 to S phase transition and inducing apoptosis.
USP14 , one member of the ubiquitin-specific proteases DUBs family , is associated with the proteasome complex and inhibits proteolysis by catalyzing protein deubiquitination ( 14 ) .
Although present in Xenopus extracts (N.V.D., R.W.K., unpublished data), levels of USP14 associated with proteasomes in extract may be insufficient to impede proteolysis.
These studies further confirmed two observations noted independently, i.e., (1) that inhibition of USP14 up-regulates proteasomal proteolysis in the cells from young donors, perhaps by virtue of 20S gate opening, but fail to do so in cells from the elderly, and (2) that inhibition of USP14 in T cells from both young and elderly donors influences the levels of poly-ubiquitinated proteins and free ubiquitin.
Small molecule inhibitor of USP14, IU1, enhances proteasomal proteolysis of ubiquitinated substrate in T cells obtained from young but not those from elderly donors.
In contrast, RNAi of either UCHL5 or USP14 alone did not affect cell growth, proteasome structure, or proteolytic capacity, but increased the rate of protein degradation.
Mouse embryonic fibroblasts lacking USP14 show enhanced clearance of several disease related proteins, including tau and polyglutamine expanded ataxin-3, and overexpression of catalytically inactive USP14 increases protein degradation [XREF_BIBR].
Indeed, an inhibitor of the deubiquitinating enzyme Usp14 enhances proteasomal protein degradation and leads to the clearance of protein aggregates in human cells [XREF_BIBR].
USP14 inhibition both by either drug like IU1 or siRNA silencing has been confirmed to accelerate protein degradation XREF_BIBR XREF_BIBR, which has also been confirmed in this report; USP14 silencing also affected CuPT induced GFPu protein degradation.
Thus, Usp14 and Ubp6 appears to suppress protein degradation through three mechanisms : deubiquitination of proteasome substrates, the " noncatalytic effect, " which operates directly on the proteasom[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
We found that a selective small-molecule inhibitor of USP14 stimulates protein degradation in vitro and in cells, and also attenuates toxic effects of oxidatively damaged proteins.
We also found enhanced expression of transcripts encoding two ubiquitin specific proteases, including USP14 that suppresses protein degradation through deubiquitination of proteasome substrates, non catalytic inhibition of proteasome activity, and by regulating autophagy.
The RNAi of UCHL5 or USP14 alone does not affect cell growth and proteasome composition but accelerates cellular protein degradation; however, RNAi of both UCHL5 and USP14 can inhibit cellular protein degradation.
The ability of USP14 to prevail over the proteasome in a kinetic competition may explain the suppression of protein degradation by USP14 's deubiquitinating activity 3.
Other inhibitors of the ubiquitin-proteasome system such as highly specific inhibitors of the proteasome associated deubiquitinating enzyme Usp14 could also show efficacy against aneuploid cells and thus could be used in the treatment of aneuploid cancers.
Furthermore, since deubiquitinating enzymes associated with the proteasome are responsible for ubiquitin trimming from substrates targeted to the proteasome for degradation, and in light of growing evidence that the manner in which proteasome associated deubiquitinating enzymes, USP14 and UCH37, deubiquitinate substrates can in fact suppress and delay degradation and modulate proteasome function, we decided to next analyze the functional activity of the proteasome associated deubiquitinating enzyme USP14.
The proteasome associated deubiquitinating enzyme Usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions.
Additional targets which play a role in WM cell survival include TLR7, 8 and 9, proteasome associated deubiquitinating enzymes (USP14 and UCHL5), XPO1 and CRM1 and AURKA.
In ataxia (ax J) mice, profound neurological and synaptic defects result from a loss-of-function mutation in the proteasome associated deubiquitinating enzyme Usp14, which is required for recycling ubiquitin from proteasomal substrates.
The proteasome associated deubiquitinating enzyme USP14 on one hand is functionally coupled with proteasomal activity and on the other hand a critical regulator of synaptic plasticity XREF_BIBR.
ISG15 conjugation may also help direct modified proteins to the proteasome, because the proteasome-associated deubiquitinating enzyme (DUB), USP14, can recognize ISG15-modified substrates, or it may function to attract components of the ubiquitin conjugation machinery.ISG15 plays a role in resistance to Ebola virus (also VSV and rabies virus) by blocking the activity of Nedd4 via ISGylation (reviewed in Sadler and Williams, 2008).
We report here that a small molecule selected for its capacity to inhibit the proteasome associated deubiquitinating enzyme Usp14 strongly enhances substrate degradation by the proteasome in cells.
A recent and exciting study for example explored the proteasome associated deubiquitinating enzyme USP14 as a target for proteasome enhancement [XREF_BIBR].
Still, USP14 activity can be remarkably enhanced by association with the proteasome -- up to ~ 800-fold -- suggesting its major regulatory role in proteasome function [XREF_BIBR, XREF_BIBR, XREF_BIBR].
This suggests that a unique negative feedback model, in which USP14 is activated by proteasome and then proteasomal degradation is suppressed by deubiquitination, is the basis by which USP14 serves as a critical inhibitory component of the proteasome [ xref , xref , xref ].
Indeed, phosphorylation of Usp14 (or the use of a Usp14 phosphomimetic) results in increased activity in assays with the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), for both Usp[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The resolution of this conundrum required the identification of a preferred substrate of Usp14 and of the properties that govern substrate preference.Cyclin B, ubiquitinated by its physiological ligas[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
This suggests that a unique negative feedback model, in which USP14 is activated by proteasome and then proteasomal degradation is suppressed by deubiquitination, is the basis by which USP14 serves as a critical inhibitory component of the proteasome [XREF_BIBR, XREF_BIBR, XREF_BIBR].
These results indicate that USP14 aptamer-induced proteasome activation might protect cells under various stressful conditions, including neurodegeneration.
This is consistent with previous studies showing that PGJ2 lowers 26S proteasome levels and activity [XREF_BIBR, XREF_BIBR], and inhibits some of the thiol deubiquitinases including UCH-L1 and UCH-L3 [XREF_BIBR, XREF_BIBR], as well as Ub-isopeptidase activity [XREF_BIBR], but not USP14 as shown here in our current studies.
Activity probe assays with either the whole 26S proteasome or the 19S regulatory particle showed that the compound blocked the reaction of both USP14 and UCHL5 with haemagglutinin (HA)-tagged ubiquitin vinyl methyl sulfone (VMS).
Inhibition of USP14 by a small moleculeenhances proteasome activity and decreases misfolded proteins in mammalian cells following proteotoxic stress 11.
Here we show that upon proteasome inhibition or expression of the mutant W58A USP14, association of USP14 with the 19S regulatory particle is disrupted.
Nevertheless, loss of USP14 reduced the volume of nuclear AR under DHT stimulation, which may result from the reduction of cellular AR after USP14 silencing.
In addition, western blot analysis and immunofluorescent staining assay indicated that USP14 silencing significantly downregulated the abundance of AR in both the nucleus and cytoplasm under androgen stimulation, suggesting that cytosolic USP14 is not required for AR translocation and that USP14 silencing induced decrease of nuclear AR could be due to the decrease of total AR protein.
Collectively, the results show that USP14 regulates the total AR level but not AR translocation, and mediates the responsiveness of AR + / ER - breast cancer to androgen.
Our gene expression analyses of androgen responsive prostate cancer cells exposed to IU1 or USP14 siRNA show a downregulation of PSA but not AR mRNA expression, suggesting that USP14 might not enhance the transcriptional activity of AR.
We found that dual inhibitors of USP14 and UCHL5 such as b-AP15 [XREF_BIBR] and auranofin [XREF_BIBR] reduced AR protein level in both prostate and breast cancer cells.
We found that similar to USP14 shRNA, inhibition of USP14 by IU1 also significantly decreased AR protein level in breast cancer cells, supporting the conclusion that proteasomal DUB USP14 regulates the expression of AR in breast cancer cells.
In the rescue experiments, IU1 induced AR downregulation can be reversed by proteasome inhibitor bortezomib (Velcade), suggesting that reduction of AR protein levels by the inhibition of USP14 depends on proteasome activity.
Loss of USP14 expression and function dramatically decreased AR level, blocked G 0 / G 1 to S phase transition, and triggered cell apoptosis in AR + breast cancer cells, suggesting that targeting USP14/AR axis could be a potential strategy for TNBC therapy.
Indeed, both pharmacological and genetic inhibition of USP14 markedly reduced, and conversely USP14 overexpression increased, the steady state protein levels of AR and its target gene PSA in LNcap cells (XREF_FIG).
We found that both IU1 and USP14 siRNA dramatically decreased the PSA mRNA but not the AR mRNA, indicating that USP14 inhibition or gene silence does not affect the transcription of AR (XREF_FIG).
We found that IU1 and USP14 knockdown dramatically increased the ubiquitinated AR (XREF_FIG), suggesting that USP14 is a DUB for AR, capable of reversing the ubiquitination of AR and thereby stabilizing AR proteins.
In conclusion, the current investigation represents a new mechanism by which inhibition of USP14 triggers autophagy via ER stress mediated UPR in A549 cells.
According to this observation, it was shown that USP14 suppresses the activity of Beclin1 complex and induction of autophagy by interacting with and controlling K63- rather than K48 linked ubiquitin chains of Beclin1 [XREF_BIBR].
Besides, the inhibition of USP14 by IU1-47 induced an increase of the autophagy flux, consistent with the increased degradation rate of Tau [XREF_BIBR].
In addition, it has recently been reported that USP14 negatively regulates autophagy 34, which plays an important role in the regulation of hepatic TG metabolism.
Moreover, we have for the first time demonstrated that the USP14 inhibition induces ER stress mediated autophagy in A549 cells by activation of c-Jun N-terminal kinase 1 (JNK1).
Knockdown of USP14 or its inhibition with the inhibitor IU1 (see below Section 4.1) induces the activation of autophagy, indicating that USP14 is a negative regulator of autophagy in H4 (neuroglioma) cells.
The authors ascribed these phenotypes to TRIM14 's role in promoting cGAS stabilization and provide evidence that loss of TRIM14 allows for cGAS degradation via the E3 ligase USP14, which targets cGAS to p62 dependent selective autophagy.
In addition, silencing USP14 expression with siRNA or stable expression of shRNA also caused G0/G1 cell cycle arrest (XREF_FIG), indicating that USP14 promotes G1-S transition in androgen responsive prostate cancer cells.
We confirmed that AR was highly expressed in the androgen responsive prostate cancer cells (LNcap cells) but was hardly detectable in the androgen-irresponsive prostate cancer cells (DU145 and PC3 cells) tested here (XREF_FIG), implying that the induction of cell cycle arrest by USP14 inhibition is AR dependent.
It has been reported that USP14 expression was specifically upregulated in both lung adenocarcinoma cell lines and tumor tissues , and knockdown of USP14 expression significantly inhibited cell growth and cell cycle arrest in NSCLC cells12 .
Furthermore, changes in key cell cycle regulators induced by the manipulation of USP14 function also support the notion that AR is a key target for USP14 in the prostate cancer cells.
It has been reported that USP14 expression was specifically upregulated in both lung adenocarcinoma cell lines and tumor tissues, and knockdown of USP14 expression significantly inhibited cell growth and cell cycle arrest in NSCLC cells XREF_BIBR.
It has been shown that USP14 modulates levels of key cell cycle regulatory proteins whose dysregulation is expected to affect the cell cycle [XREF_BIBR].
To investigate the molecular mechanism by which USP14 promotes cell cycle, we performed western blot to detect several key proteins that are associated with G1-S phase transition.
To explore the underlying mechanism by which USP14 promotes cell proliferation in LNcap cells, we monitored the cell cycle progression of each group exposed to various concentrations of IU1 (25, 50, 100muM) and found that inhibition of USP14 activity dramatically induced G0/G1 cell cycle arrest at different time points (0, 6, 12, 24, 48h) (XREF_FIG).
USP14 knockdown or treatment with USP14 inhibitor IU1 induced G0/G1 cell cycle arrest and suppressed cell proliferation in AR-positive and ER-negative breast cancer cells and androgen responsive prost[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Thus, Akt not only activates USP14 by a different mechanism than the proteasome, but it can cooperate with the proteasome to achieve more aggressive removal of ubiquitin from proteasome-docked substrates.
The notion that USP14 may serve as a focal point for regulating proteasome activity was consistent with a recent report that the kinase Akt activates USP14 via phosphorylation 35.
Therefore, it remains unclear whether Ser432 phosphorylation generates a proteasome independent active form of Usp14.The activation of Usp14 by AKT may provide a mechanism whereby growth signals can s[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The notion that USP14 may serve as a focal point for regulating proteasome activity was consistent with a recent report that the kinase Akt activates USP14 via phosphorylation xref .
Since USP14 is a negative regulator of the UPS and we found USP14 can be phosphorylated and activated by Akt, we reasoned that Akt mediated activation of USP14 might lead to inhibition of the UPS and generally enhance the stability of many proteins.
However, inhibition of both FASN and USP14 had no significant synergistic effect on cancer cell proliferation and, surprisingly, it was confirmed that USP14 negatively regulates the protein level and activity of FASN in cancer cells.
In LNCaP, MCF7 (human breast cancer cell), and A549 cell (human lung cancer cell), endogenous FASN protein levels were increased by USP14 knockdown contrary to the results in MPHs (Figure 2a).
In future studies, the investigation of alterations in the transduction pathway of USP14 and FASN using transcriptomic analysis is warranted to understand not only the interaction between the two proteins but also the correlation of the interactomes.Taken together, the results reveal that USP14 negatively regulates FASN levels unexpectedly in the cancer cells, and as a result, inhibition of USP14 was not conducive to cancer cell death through inhibition of FASN.
Based on a previous finding that Usp14 is a novel DUB for FASN and enhances FASN stability by blocking proteasomal degradation in MPHs, we expected that a USP14 inhibitor IU1 could further reduce the activity of FASN by interfering with FASN stability when used together with a FASN inhibitor.
In addition to USP2a, which is well known as a DUB that regulates the level of FASN, a recent study showed that Usp14 significantly contributes to the development of hepatosteatosis by maintaining the stability of FASN in MPHs [7,20].
As a result, USP14 overexpression reduced enzymatic activity of FASN, whereas USP14 deficiency significantly increased FASN activity in cancer cells (Figure 3a,b).
Moreover, overexpression of USP14 in MM cell adhesion model could enhance the ability of cell adhesion by regulating Wnt signaling pathways, thereby promoting the CAM-DR in MM.
These data suggest that knockdown of USP14 inhibits the proliferation and tumorigenesis in ESCC cells by suppressing and inhibiting the Wnt and beta-catenin signaling pathway.
Depletion of Usp14 attenuated downstream Wnt signalling which was further evidenced when correlation between the levels of Usp14 and beta-catenin in colon tissues was observed.
These results suggest that USP14 specifically enhanced NLRC5 mediated inhibition of NF-kappaB activation through the inhibition of NLRC5 ubiquitination via its DUB activity.
Because USP14 specifically enhanced NLRC5 mediated inhibition of NF-kappaB activation, we reasoned that USP14 deficiency would result in the accumulation of ubiquitinated NLRC5 and increased NF-kappaB activation under physiological conditions.
In this study, we aimed to clarify the role of the USP14-NLRC5 pathway in wear particle induced osteolysis in vitro and in vivo We found that NLRC5 or USP14 overexpression inhibits titanium particle induced proinflammatory tumor necrosis factor alpha (TNF-alpha) production and NF-kappaB pathway activation, and also decreases M1 macrophage polarization and PI3K and AKT pathway activation.
Conversely, USP14 KD THP-1 cells clearly enhanced NF-kappaB activation and the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-1beta.
Based on these results, USP14 can induce the activation of NF-kappaB and regulate the up-regulation of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and interleukin-18 (IL-18) expression by activating the NF-kappaB signal pathway.
Because USP14 specifically enhanced NLRC5 mediated inhibition of NF-kappaB activation, we reasoned that USP14 deficiency would result in the accumulation of ubiquitinated NLRC5 and increased NF-kappaB activation under physiological conditions.
These results suggest that USP14 specifically enhanced NLRC5 mediated inhibition of NF-kappaB activation through the inhibition of NLRC5 ubiquitination via its DUB activity.
Higher intrinsic USP14 levels in BMMs compared with BMDCs " forced " BMMs to move toward a state of higher USP14 values and subtly raised the sensitivity to NLRC5 in BMMs.
In contrast, ubiquitin specific protease 14 (USP14) specifically removes the polyubiquitin chains from NOD like receptor family CARD domain containing 5 (NLRC5) and thereby enhances the NLRC5 mediated inhibition of NF-kappaB signaling.
USP14 deficiency significantly attenuates the inhibitory ability of NLRC5 on NF-kappaB activation, whereas the K1178R NLRC5 mutant showed similar inhibition of NF-kappaB activity in both WT and USP14 KO cells.
PtPT is also strikingly distinct from IU1, a USP14 inhibitor reported previously [ xref ], which inhibits USP14 but not UCHL5 activity and promotes the degradation of ubiquitinated proteins.
We found that (i) USP14 could bind to AR, and additionally, both genetic and pharmacological inhibition of USP14 accelerated the ubiquitination and degradation of AR; (ii) downregulation or inhibition of USP14 suppressed cell proliferation and colony formation of LNcap cells and, conversely, overexpression of USP14 promoted the proliferation; and (iii) reduction or inhibition of USP14 induced G0/G1 phase arrest in LNcap prostate cancer cells.
Furthermore, we found that NCOA4 was upregulated by ubiquitin specific peptidase 14 (USP14) via a deubiquitination process in damaged neurons, and we found evidence of pharmacological inhibition of USP14 effectively reducing NCOA4 levels to protect neurons from ferritinophagy-mediated ferroptosis.
Another example of neuroprotection was found in stroke, in which decreased expression of USP14 by microRNA or miR-124, as well as USP14 inactivation, was linked to neuron survival in the post-ischemic mouse brain 69.
11) strongly reduced the deubiquitinase activity in the control lysates, and rendered TRIM11 ineffective in reducing deubiquitinase activity, indicating that TRIM11 specifically inhibits the deubiquitinase activity of USP14.
In contrast, the amounts of USP14 were decreased in the pellets and conversely increased in the detergent-soluble SN, consistent with its displacement from the proteasome by TRIM11.
It has been reported that USP14 expression was specifically upregulated in both lung adenocarcinoma cell lines and tumor tissues, and knockdown of USP14 expression significantly inhibited cell growth and cell cycle arrest in NSCLC cells XREF_BIBR.
As shown in XREF_FIG, USP14 inhibition or silence failed to induce apoptosis or PARP cleavage but instead induced moderate decreases of p53 and Bax, suggesting that cell growth suppression mediated by USP14 inhibition or silence is through promoting cell proliferation, independent of cell death.
USP14 inhibition via administration of IU1 or USP14 specific siRNA and shRNA enhanced cell growth inhibition and apoptosis induction by enzalutamide in breast cancer cell lines in vitro and in vivo.
Since we have observed that inhibition or knockdown of USP14 inhibited cell growth in LNcap cells, we further investigated whether USP14 inhibition or silence induces cell death of LNcap cells by measuring Annexin V-FITC and PI-positive cells with flow cytometry and by measuring PARP cleavage and p53 and Bax protein expression with western blot analyses.
It has been reported that USP14 expression was specifically upregulated in both lung adenocarcinoma cell lines and tumor tissues , and knockdown of USP14 expression significantly inhibited cell growth and cell cycle arrest in NSCLC cells12 .
We previously confirmed that a specific USP14 and UCH37 inhibitor b-AP15 4 inhibited tumor cell growth and induced apoptosis and in vitro (data not shown).
Since we have observed that inhibition or knockdown of USP14 inhibited cell growth in LNcap cells, we further investigated whether USP14 inhibition or silence induces cell death of LNcap cells by measuring Annexin V-FITC/PI-positive cells with flow cytometry and by measuring PARP cleavage and p53 and Bax protein expression with western blot analyses.
To investigate the molecular mechanism by which USP14 promoted proliferation and invasion in ESCC cells, we examined the effects of USP14 on the activation of the Wnt and beta-catenin signaling pathway.
In addition, IU1, a small-molecule inhibitor of USP14, accelerated the degradation of a subset of proteasome substrates and suppressed cell proliferation, migration, and invasion in lung cancer and cervical cancer.
USP14 depletion or its specific inhibitor IU1 treatment decreased cell proliferation , invasion , migration , and Vascular Mimicry ( VM ) formation even under hypoxia conditions in HCC cell lines .
The MTT assay showed that USP14 overexpression increased the cell viability in BGC-823 and MGC-803 cells, and the knockdown of USP14 repressed the cell viability in BGC-823 and MGC-803 cells.
As shown in Figure XREF_FIG, pharmacological inhibition of USP14 caused dose dependent inhibition of cell viability in endometrial cancer cell lines with an IC 50 of 181.3 and 117.5 nM for HEC155 (left panel) and ECC1 (right panel), respectively.
The DUB USP14 suppresses turnover of Tau and TDP-43 in mouse embryonic fibroblasts (MEFs) by impairing the protea-some; therefore, small-molecule inhibitors of USP14 could help clear these toxic proteins from cells.
In conclusion, pharmacologically inhibiting (with low or high IU1 concentrations) or genetically down-regulating USP14 fail to enhance proteasomal degradation of Ub-proteins or Tau in neurons.
Intriguingly, Lee et al. demonstrated that IU1, a selective small-molecule inhibitor of USP14, accelerated proteasomal degradation of tau and TDP-43, which have been implicated in neurodegenerative diseases XREF_BIBR.
These data suggest that knockdown of USP14 inhibits the proliferation and tumorigenesis in ESCC cells by suppressing and inhibiting the Wnt and beta-catenin signaling pathway.
However, we have recently unraveled that Aur inhibits 19S proteasome associated DUBs (mainly UCHL5 and USP14), accumulates ubiquitinated proteins (Ub-prs), and induces unfolded protein response (UPR) followed by cell apoptosis.
Here we report that
(i) Aur shows proteasome-inhibitory effect that is comparable to that of
bortezomib/Velcade (Vel); (ii) different from bortezomib, Aur inhibits
proteasome-associated deubiquitinases (DUBs) UCHL5 and USP14 rather than the 20S
proteasome; (iii) inhibition of the proteasome-associated DUBs is required for
Aur-induced cytotoxicity; and (iv) Aur selectively inhibits tumor growth in
vivo and induces cytotoxicity in cancer cells from acute myeloid leukemia
patients.
We found that the remaining active forms of both UCHL5 and USP14 (i.e., those can be covalently bound by HA-UbVS) were clearly reduced in the 26S proteasomes pre-treated with Aur at 2 muM and became completely undetectable in those pre-treated with 40 muM Aur, indicating that Aur inhibits both UCHL5 and USP14.
Here we report that (i) Aur shows proteasome-inhibitory effect that is comparable to that of bortezomib and Velcade (Vel); (ii) different from bortezomib, Aur inhibits proteasome associated deubiquitinases (DUBs) UCHL5 and USP14 rather than the 20S proteasome; (iii) inhibition of the proteasome associated DUBs is required for Aur induced cytotoxicity; and (iv) Aur selectively inhibits tumor growth in vivo and induces cytotoxicity in cancer cells from acute myeloid leukemia patients.
We found that the remaining active forms of both UCHL5 and USP14
(i.e., those can be covalently bound by HA-UbVS) were clearly reduced in the 26S
proteasomes pre-treated with Aur at 2 μM and became completely undetectable in
those pre-treated with 40 μM Aur (Fig. xref ), indicating that Aur inhibits both UCHL5 and USP14.
30 Overall, these data suggest that increased expression of Usp14 may enhance beta-catenin-mediated transformation of normal colon cells, but not metastasis of malignant colon cancer cells.
In contrast, studies using OSCC cells have demonstrated that TGF-beta signals can mediate Epithelial- mesenchymal transitions, further contributing to cancer cells migration and invasion (Meng et al.,[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
For example, Zhu et al. reported that expression of USP14 was increased in breast cancer tissues, and downregulation of USP14 significantly inhibited breast cancer cell proliferation and metastasis XREF_BIBR.
Over-expression of a catalytically inactive form of USP14 rescues the PPF deficit and restores synaptic vesicle number, indicating that USP14 regulates presynaptic structure and function independently of its role in deubiquitination.
To determine whether the BALBnmf375 mice were also resistant to the PPF deficit caused by USP14 deficiency in the C57ax J mice, we analyzed PPF in the CA1 region of the nmf375 hippocampus.
Finally, the PPF deficit caused by loss of USP14 can be rescued by pharmacological inhibition of proteasome activity, suggesting that inappropriate protein degradation underlies the PPF impairment.