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.
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.
Our current study showed that IU1, a pharmacological deubiquitinating enzyme USP14 selective inhibitor, dramatically decreased MDM2 level, blocked G0/G1 to S phase transition, decreased cell growth and triggered cell apoptosis in cervical cancer cells, suggesting that targeting USP14 and MDM2 axis could be a potential strategy for cervical cancer therapy.
We further detected the change of MDM2-AR interaction using co-immunoprecipitation (co-IP), and found that USP14 inhibition or silence increased the binding of MDM2 to AR (XREF_FIG), suggesting that USP14 inhibits the degradation of AR by decreasing the expression and phosphorylation of MDM2.
Auranofin (Aur) inhibits proteasome associated deubiquitinases (DUBs) UCHL5 and USP14 rather than the 20S proteasome; inhibition of the proteasome associated DUBs is required for Aur induced cytotoxicity; and Aur selectively inhibits tumor growth in vivo and induces cytotoxicity in cancer cells from patients with acute myeloid leukemia [XREF_BIBR].
Ctrl and USP14 KD THP-1 cells were treated with or without LPS for different time periods and the activation of TLR4 downstream signaling molecules was analyzed by western blotting.
To verify such results, Ctrl or USP14 KD THP-1 cells were treated with or without LPS and then NF-kappaB activation was assessed by using p65- or p50-DNA binding assay.
We herein found that YTHDF1 could promote USP14 protein translation in a m 6 A dependent manner and USP14 overexpression reversed the tumor suppressive effects caused by YTHDF1 knockdown in GC cells.
We found that knockdown of YTHDF1 decreased the protein abundance of USP14 rather than its transcription levels in AGS and BGC-823 cells as expected by RT-qPCR and western blotting analysis (XREF_FIG).
Collectively, a better understanding about the regulation of PrP Sc clearance caused by USP14 might contribute greatly to the development of therapeutic strategies for prion diseases.
Results demonstrated that an inhibitor of USP14 reduced PrP C in mouse neuroblastoma cells, as well as PrP Sc, indicating that USP14 negatively regulates degradation of prion protein.
As shown in XREF_FIG, overexpression of HA-USP14 (confirmed in the accompanying SDS-PAGE) increased steady state levels of CXCR4 and also blocked CXCL12 evoked down-regulation of CXCR4 levels characteristic of control cells (vector alone).
As shown in XREF_FIG, overexpression of HA-USP14 (confirmed in the accompanying SDS-PAGE) increased steady state levels of CXCR4 and also blocked CXCL12 evoked down-regulation of CXCR4 levels characteristic of control cells (vector alone).
In summary, our findings demonstrated a novel mechanism by which USP14 negatively regulated prion protein degradation via the proteasome, although the detailed mechanism remains unclear.
Results demonstrated that an inhibitor of USP14 reduced PrP C in mouse neuroblastoma cells, as well as PrP Sc, indicating that USP14 negatively regulates degradation of prion protein.
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.
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].
Although the role of USP14 can not be completely ruled out as a proteasome associated DUB enzyme that can affect the expression of TRAF6, these results suggest that USP14 inhibits the interaction of Beclin 1 to TRAF6 through the competitive interaction to TRAF6 with Beclin 1.
CXCL12, a CXCR4 agonist, induces a time dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family.
Intriguingly, Ub deficiency induced the upregulation of USP14 in mammals (Ryu KY, unpublished data) and Ubp6 in yeast XREF_BIBR, thereby sparing Ub from proteasomal degradation and increasing levels of free Ub in an attempt to reach Ub homeostasis.
Interestingly, TgT suppressed the effects of MEHP on chemotaxis in adult neutrophils, suggesting that that the role of PPAR-gamma signaling in chemotaxis is developmentally regulated.
The PPAR-gamma agonist, TgT, reduced fMLP induced chemotaxis in both control and MEHP treated adult neutrophils, but had no significant effects on neonatal cells.
USP14 knockdown significantly enhanced RIG-I-triggered type I IFN signaling and inhibited VSV replication both in mouse peritoneal macrophages and THP1 cells.
IFN I induced expression of TRIM14 favors the recruitment of USP14, a deubiquitinase which in turn promotes further IFN I expression by modifying and protecting cGAS from autophagy mediated degradation; thus, Trim14 -/- mice are more susceptible to infection.
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.
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.
Co-expression of Flag tagged wild-type (WT) USP14, but not catalytically inactive (CI) USP14 31 or GFP, significantly increased the FASN half-life, further supporting the notion that USP14 could stabilize FASN protein.
Notably, protein expression of the three key enzymes in the fatty acid synthesis pathway, ACLY, ACACA, FASN, and seven enzymes in the fatty acid degradation pathway were downregulated, which further indicated a possible role of USP14 in fatty acid metabolism.
As a result, knockdown of endogenous FASN expression largely blocked the roles of USP14 overexpression, suggesting that the role of USP14 in the regulation of hepatic TG metabolism is, at least in part, dependent on upregulation of FASN.
Previous studies have shown that downregulation of USP14 leads to increased expression of E-cadherin and decreased expression of N-cadherin and vimentin in esophageal squamous cell carcinoma (ESCC) cells, as well as suppresses the migration and invasion of ESCC cells through the Wnt and beta-catenin signaling pathway.
The analysis of the ubiquitination of vimentin revealed that increased USP14 decreased the levels of ubiquitinated vimentin, and the knockdown of USP14 led to an increase in the levels of ubiquitinated vimentin.
A recent study revealed that USP14 knockdown inhibited the migration of breast cancer cells by increasing the expression of vimentin and reducing the expression of E-cadherin, suggesting that USP14 might enhance the metastatic ability of breast cancer cells by initiating the epithelial-mesenchymal transition process.20 However, whether USP14 promotes ESCC metastasis through the same mechanism or other pathways remains unclear.
Similarly, knockdown of UCH37 and USP14 or b-AP15 treatment rescued p53 protein, induced by COPS5 overexpression, and enhanced the activity of transfected luciferase reporter plasmids for p53, Bax, and p21 expression and protein levels of p53 downstream target genes BAX and p21.
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.
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.
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).
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).
Mechanistically, sustained ER stress up-regulated the deubiquitinating enzyme ubiquitin specific peptidase 14 (USP14), which increased the stability and levels of 3 ',5 '-cyclic monophosphate responsive element binding (CREB) protein (CBP) to enhance glucagon action and hepatic gluconeogenesis.
Studies have shown that sustained ER stress upregulates USP14 from the transcription level through ATF4, thus improving the stability and level of CREB binding protein CBP, enhancing the role of glucagon and hepatic gluconeogenesis.
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.
USP14 inhibition via gene knockdown or small molecules induces cell death in breast cancer ( 24 ) , acute myeloid leukemia ( 25 ) , and diffuse large B-cell lymphoma ( 26 ) .
Moreover, inhibition or silencing of USP14 dramatically induced higher levels of cell death and PARP cleavage in MDA-MB 231 and MDA-MB 453 than in MCF7 breast cancer cells.
This finding is consistent with another study demonstrating that the inhibition of USP14 increases IkappaB stability in rat lungs and prevents ventilator induced lung injury.
This was also confirmed by computational molecular docking, K48 linked polyubiquitin disassembly, and HA-UbVS competitive binding assay; these experiments showed that AF might inhibit the proteasomal cysteine DUBs UCHL5 and USP14.
Interestingly, we found that different from bortezomib, AF inhibits 19S proteasome-associated DUBs UCHL5 and USP14 but not the 20S proteasome activity.
Interestingly, we found that different from bortezomib, AF inhibits 19S proteasome associated DUBs UCHL5 and USP14 but not the 20S proteasome activity.
Another compound, copper pyrithione (CuPT), was reported to target both 19S proteasome specific DUBs, UCH37 and USP14, as well as 20S proteolytic peptidases.
Here we report that zinc pyrithione (ZnPT) targets the proteasome associated DUBs (USP14 and UCHL5) and inhibits their activities, resulting in a rapid accumulation of protein-ubiquitin conjugates, but without inhibiting the proteolytic activities of 20S proteasomes.
And only recently we have definitely confirmed that nickel pyrithione (NiPT) inhibits the 19S proteasome-associated deubiquitinases (DUBs) USP14 and UCHL5, but not the 20S proteasome peptidases, and the inhibition of proteasome-associated DUBs induces NiPT-mediated cytotoxicity, revealing a novel mechanism for the anti-cancer effects of nickel-containing compounds [ xref ].
Intriguingly, Usp14 binds the alpha1 GABA A receptor subunit, and ax J mice lacking Usp14 exhibit increased GABA A receptor levels at Purkinje cell-surface membranes and increased inhibitory postsynaptic currents (IPSCs), which suggest that ubiquitin dependent GABA A receptor turnover at cerebellar synapses contributes to behavioral impairment.
Colocalization experiments and in vitro binding studies further indicated that USP14 and GABA A receptors are found together at synapses, that the C-terminus of USP14 can interact with the alpha1 loop of the GABA A receptor, and that expression of a GABA receptor peptide that binds USP14 can promote expression of surface GABA A receptors in HEK cells [XREF_BIBR].
Together, these data demonstrate physical interaction of GABA A R alpha1 and the ubiquitin specific protease Usp14, and suggest that the observed GABA A R redistribution in ataxia mice (XREF_FIG and XREF_FIG) is directly caused by the loss of Usp14, thereby indicating that GABA A R turnover is ubiquitin dependent.
At the same time, liver specific knockout of USP14 eliminated the effect of ER stress on glucose metabolism and also improved hyperglycemia and glucose intolerance in obese mice.
Recently, researches have revealed USP14 enhances cisplatin resistance through affecting Akt and ERK signaling pathways and accelerates cell proliferation and migration in GC.
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.
Moreover, inhibition or silencing of USP14 dramatically induced higher levels of cell death and PARP cleavage in MDA-MB 231 and MDA-MB 453 than in MCF7 breast cancer cells.
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.
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.
Similarly, knockdown of UCH37 and USP14 or b-AP15 treatment rescued p53 protein, induced by COPS5 overexpression, and enhanced the activity of transfected luciferase reporter plasmids for p53, Bax, and p21 expression and protein levels of p53 downstream target genes BAX and p21.
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.
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.
Notwithstanding this success, the potency of pimozide (IC 50 ~ 2muM) is lower than those of clinically approved UPS inhibitor 26S proteasome inhibitor Bortezomib (IC 50 ~ 100nM) (XREF_FIG and XREF_TABLE), and another UPS inhibitor VLX1570, which inhibited the DUBs UCHL5 and USP14 (IC 50 ~ 100nM) and was previously studied in clinical trials but terminated due to limiting toxicities (study identifier NCT02372240).
Notwithstanding this success, the potency of pimozide (IC50 ~2 μM) is lower than those of clinically approved UPS inhibitor 26S proteasome inhibitor Bortezomib (IC50 ~100 nM) (Fig. 1 and Table 2), and another UPS inhibitor VLX1570, which inhibited the DUBs UCHL5 and USP14 (IC50 ~100 nM) and was previously studied in clinical trials but terminated due to limiting toxicities (study identifier NCT02372240) (ClinicalTrials.gov, 2018; Wang et al., 2016).
A study by Doeppner et al. (2013) found that miR-124 reduces expression of the target deubiquitinating enzyme Usp14, thereby increasing repressor-element-1-silencing transcription factor degradation.
Although the -10 region is less conserved, there is a TGT motif one base upstream of the -10 region which is known to enhance transcription XREF_BIBR, XREF_BIBR.
Knockdown or inhibition of USP14 significantly abrogated hypoxia and reoxygenation induced upregulation of nuclear factor kappa B (NF-kappaB) activation and proinflammatory cytokine production.
Knockdown of YTHDF1 suppressed cell growth and colony formation, while overexpression of USP14 could rescue sh-YTHDF1 expressing GC cells from these effects (XREF_SUPPLEMENTARY).
They further demonstrated that inhibition of USP14 not only promoted the degradation of classic substrates of the UPP, but also promoted the clearance oxidized proteins and enhanced the resistance to oxidative stress [XREF_BIBR], indicating that the UPP is involved in targeting oxidized proteins for degradation.
They further demonstrated that inhibition of USP14 not only promoted the degradation of classic substrates of the UPP, but also promoted the clearance oxidized proteins and enhanced the resistance to oxidative stress [XREF_BIBR], indicating that ubiquitination is involved in targeting oxidized proteins for degradation.
By using overexpression model of chicken UCH-L5 and b-AP15 inhibitor of UCH-L5 and USP14 [XREF_BIBR], which inhibited UCH-L5 at low molar concentration, but it did not significantly inhibit other DUBs in HD11 cells (XREF_SUPPLEMENTARY), we observed the overexpression of UCH-L5 had a negative effect on cell viability, which depended on its catalytic activity as a DUB, since if UCH-L5 overexpressing cells were treated with b-AP15 inhibitor, the cell viability was partially restored (XREF_FIG).
We also found that inhibition of USP-14 and UCHL5 activities by the ITCs caused increased levels of USP14 and UCHL5 proteins, but not the third 19S deubiquitinating enzyme (DUB), POH1 and RPN11, suggesting feedback loop activation and further supporting that ITCs are inhibitors of proteasomal cysteine DUBs.
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.
Interestingly, it appears USP14 not only negatively regulates the UPS, but also negatively regulates autophagy through removing Lys63 linked ubiquitin chains from the autophagy regulator Beclin-1 [XREF_BIBR].
The resulting conjugates were incubated with purified human proteasomes that were washed with high salt to eliminate USP14, a deubiquitinating enzyme that can antagonize cyclin B1 degradation in vitro XREF_BIBR, XREF_BIBR.
Furthermore, we revealed a previously uncharacterized biological function of USP14 : Inhibition of USP14 reduced the stability of CBP, thereby lessening the severity of pathogen induced lung inflammation.
The resulting conjugates were incubated with purified human proteasomes that were washed with high salt to eliminate USP14, a deubiquitinating enzyme that can antagonize cyclin B1 degradation in vitro XREF_BIBR, XREF_BIBR.
A series of studies have demonstrated that Ubp6 and USP14 enhances the ATPase activity of proteasome and opens the 20S gate upon treatment with ubiquitin, ubiquitin protein conjugate or active site directed probes, such as ubiquitin aldehyde (Ubal) or ubiquitin vinyl sulfone (UbVS) (XREF_FIG A, right bottom panel) [XREF_BIBR, XREF_BIBR, XREF_BIBR, XREF_BIBR].
To determine whether the role of USP14 is dependent on insulin, C57BL/6 were treated with streptozotocin (STZ), which induced insulin deficiency due to the selective pancreatic beta-cell toxicity.
Notwithstanding this success, the potency of pimozide (IC 50~2 μM) is lower than those of clinically approved UPS inhibitor 26S proteasome inhibitor Bortezomib (IC 50~1 00 nM) ( Fig. 1 and Table 2 ), and another UPS inhibitor VLX1570, which inhibited the DUBs UCHL5 and USP14 (IC 50~1 00 nM) and was previously studied in clinical trials but terminated due to limiting toxicities (study identifier NCT02372240) (ClinicalTrials.gov, 2018; Wang et al., 2016) .
Notwithstanding this success, the potency of pimozide (IC50 ~2 μM) is lower than those of clinically approved UPS inhibitor 26S proteasome inhibitor Bortezomib (IC50 ~100 nM) (Fig. 1 and Table 2), and another UPS inhibitor VLX1570, which inhibited the DUBs UCHL5 and USP14 (IC50 ~100 nM) and was previously studied in clinical trials but terminated due to limiting toxicities (study identifier NCT02372240) (ClinicalTrials.gov, 2018; Wang et al., 2016).
We assessed the protective efficacy of inhibiting or downregulating USP14 in rat and mouse (Usp14 axJ) neuronal cultures treated with prostaglandin J2 (PGJ2).
Accordingly, this unexpected restriction did not allow us to figure out whether " wild type " Tgt, Tgt (Cys158Val) and Tgt (Val233Gly) were indeed able to accept queuine as a substrate nor did it allow us to determine Michaelis-Menten parameters as an indicator for affinity and catalytic activity.
Accordingly, this unexpected restriction did not allow us to figure out whether " wild type " Tgt, Tgt (Cys158Val) and Tgt (Val233Gly) were indeed able to accept queuine as a substrate nor did it allow us to determine Michaelis-Menten parameters as an indicator for affinity and catalytic activity.
Likewise, studies with cortical cultures from Usp14 axJ mice [XREF_BIBR] which exhibit a 90-95% loss of USP14 (XREF_FIG, panel 1), show that USP14 depletion failed to prevent the deleterious effects of PGJ2 tested, i.e. caspase activation and accumulation of Ub-proteins (XREF_FIG, panels 2 and 3, respectively).
USP14 inhibited the degradation of YFP-CL1, as overexpression of USP14 increased YFP-CL1 abundance, while silencing USP14 accelerated YFP-CL1 degradation.
USP14 inhibited the degradation of YFP-CL1, as overexpression of USP14 increased YFP-CL1 abundance, while silencing USP14 accelerated YFP-CL1 degradation.
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.
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.
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]
High proportions (> or = 50%) of GGT --> TGT mutations from B [a] P, B [b] F and 5MC induced tumors and GGT --> CGT mutations from CPP tumors were observed and were statistically significant compared to mutations in tricaprylin control tumors.
The results of the Western blot indicated that downregulation of USP14 increased the expression of epithelial marker E-cadherin, while it decreased levels of mesenchymal markers vimentin and N-cadherin in EC109 (XREF_FIG) and TE10 cells (XREF_FIG), respectively.
A recent study revealed that USP14 knockdown inhibited the migration of breast cancer cells by increasing the expression of vimentin and reducing the expression of E-cadherin, suggesting that USP14 might enhance the metastatic ability of breast cancer cells by initiating the epithelial-mesenchymal transition process.20 However, whether USP14 promotes ESCC metastasis through the same mechanism or other pathways remains unclear.
In our previously study, we have reported the deubiquitinase USP14 promotes CD36 stability via removing its ubiquition and inhibiting USP14 significantly decreases the formation of foam cell 21.
Mutation of USP14 in mice or ataxin-3 in humans causes ataxia (Crimmins et al., 2006; Duenas et al., 2006) , whereas the S18Y allele of human UCH-L1 confers protection against sporadic Parkinson's disease.
We propose the degradation of USP14 by UPS as the underlying molecular mechanism responsible for the physiological and behavioural impairments observed (XREF_FIG).
In detail, UPS inhibition disrupts the association of USP14 with the P19S regulatory particle of the UPS, while fostering its interaction with either the autophagy protein GABARAP or the heat-shock-related co-chaperonin HSC70 [XREF_BIBR].
The - 273 to - 57 promoter region of CD36 was amplified from the DNA samples using the primer pair : forward 5 '-AGG GGA AAC TCA GCA AGT CAG T-3 ' and reverse 5 '-CAG TGT GTG GTT TGT TGC CAG AG-3 '.
When lysates of MDA-MB-231 cells treated with 25 µM of SFN were used in the Ub-VS assay, inhibition of both USP14 and UCHL5 by SFN was observed ( xref , lanes 3 vs .
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 (Fig. xref a– xref ); however USP14 knockdown did not affect the proliferation of T47D cells (Fig. xref ), which could be related to the fact that these cells express very high levels of ER and very low levels of AR (Fig. xref ).
In a recent study by Kim et al. [216], proteasome-mediated proteolysis was increased by knocking down USP14 with small interfering RNA (siRNA) which led to a significant impairment of autophagic flux.
We here explored the phenotypic response of colon cancer cells to b-AP15, a bis-benzylidine piperidone previously shown to inhibit the proteasome deubiquitinases (DUBs) USP14 and UCHL5.
This induction appears to increase the levels of Ubp6 and Usp14 on the 26S proteasome, and thus presumably increases its deubiquitinating activity and possibly also gate opening upon binding of a Ub conjugate.
Another compound, copper pyrithione (CuPT), was reported to target both 19S proteasome specific DUBs, UCH37 and USP14, as well as 20S proteolytic peptidases.
miR-34a-5p biotin labeled probe was synthesized by IDT with the probe sequence of 5 ' Biotin-TGG CAG TGT CTT AGC TGG TTG T as well as the negative control probe with the sequence of 5 ' Biotin-ACG TGA CAC GTT CGG AGA ATT.
In the denervated soleus muscle, beta-carotene administration significantly decreased the expression levels of Atrogin-1, MuRF1, USP14 and USP19 (P < 0.05, n 5) and the levels of ubiquitin conjugates.
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 .
Conversely, methylene-spaced amide substitution at the 2-position of the pyrimidine ring (50) was found to increase USP14 potency.As part of MISSION Therapeutics’ research into developing drug-like inhibitors of several DUBs, we have successfully co-crystallised an example from this fused thiophene pyrimidine series into USP14 CD and have a 3.
To determine whether DUBs of the 26S proteasome regulate proteasomal ubiquitination, we set up in vitro ubiquitination reactions in which Ub aldehyde (an inhibitor of both Usp14 and Uch37) or 1,10-phenanthroline (an inhibitor of Rpn11) was added in the reactions.
To determine whether the increase of TG retention by USP14 overexpression relies on its regulation of FASN, we carried out adenoviral shRNA mediated FASN knockdown combined with USP14 overexpression in C57BL/6 mice.
Taken together, these data indicate that functional Usp14 enhances direct Ag presentation, preferentially of DRiP derived peptides, suggesting that the processing of DRiPs is in some ways different from other forms of Ag.
At the molecular level, we find that inhibition of USP14 rapidly triggers accumulation of poly-ubiquitinated proteins and chaperones, mitochondrial dysfunction, ER stress, and a ROS production leading to a caspase independent cell death.
Moreover, overexpression of catalytically dead USP14 reduces the levels of prion aggregates in PrPC-overexpressing Neuro2a mouse neuroblastoma cells (Homma et al. 2015).
We also found enhanced expression of transcripts encoding two ubiquitin-specific proteases ( Table 2 ) , including USP14 that suppresses protein degradation through deubiquitination of proteasome substrates , non-catalytic inhibition of proteasome activity , and by regulating autophagy ( de Poot et al. 2017 ; Xu et al. 2016 ) .
Moreover, overexpression of catalytically dead USP14 reduces the levels of prion aggregates in PrPC-overexpressing Neuro2a mouse neuroblastoma cells (Homma et al. 2015).
Likewise, recombinant USP14 inhibited proteasomal peptidase activity in cell lysates, but this effect was counteracted by recombinant TRIM11 (Fig. xref ).
Herpes simplex virus (HSV-1) induces TRIM14 expression and recruits the deubiquitinating enzyme USP14, which inhibits the K48 linked polyubiquitination of cGAS to increase its stability and enhances the immune response against HSV-1.
We further demonstrate that USP14 inhibition can be therapeutically exploited to enhance sensitivity and to overcome resistance to conventional chemotherapeutic agents for treating NB .
Conversely, USP14 might also activate proteolysis by degrading ubiquitin chains on target proteins and thereby enhance gate opening of the 20S proteasome.
TGT studies exhibited increased lag time, time to peak and reduced peak thrombin generation, but no change in endogenous thrombin potential (ETP) on Day 1.
Inhibitors of deubiquitinases Usp14 and Rpn11 suppress the growth of estrogen positive breast cancer cells, which is accompanied by cell cycle arrest, apoptosis induction, and a decrease in ERalpha content [XREF_BIBR].
The data that USP14 regulated AR-V7 in our study provided new insights in USP14 function, and it corresponds with the conclusion that inhibition of USP14 can enhance enzalutamide treatment in hormone sensitive cancer cells.
The results of the meta-analysis showed that TGt could reduce 24-hour urinary protein , increase serum albumin , improve clinical efficacy , and reduce disease recurrence rate in patients ( P < 0.05 ) with CKD compared with adrenocortical hormones or immunosuppressants .
TGt could significantly reduce the level of serum creatinine (Scr) in patients with CKD (P < 0.05), but it was not significant in reducing the level of blood urea nitrogen (P> 0.05).
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).
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.
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.
USP14 inhibition causes accumulation of ubiquitinated proteins and induces unfolded protein response ( UPR ) in NB Previous studies showed that blockade of USP14 DUB activity increases intracellular ubiquitinated proteins in many cell types ( 36 , 37 ) .
The inhibition of USP14 can also increase the degree of ubiquitination of Dvl and significantly inhibit the downstream of Wnt signal transduction [ 18 ] .
USP14 impedes degradation of ubiquitinated proteins by removing ubiquitin chains from its substrates , while it could promote protein degradation via increasing proteasome activation .
diaph3 was infected by lentiviruses, respectively, containing the following plasmids : TRCN0000154182 that targets CCT TCG GAT TTA ACC TTA GCT, TRCN0000150850 that targets GCA TGA CAA GTT TGT GAC AAA, TRCN0000150903 that targets GCT CAG TGC TAT TCT CTT TAA, and TRCN0000151280 that targets CGT GTC AGA ATA GCT AAA GAA.
However, TRIM11 dependent tumor cell growth can be suppressed by USP14 overexpression in the HCT116 cell derived xenograft model, which suggests possible opposing roles of USP14 in tumorigenesis [XREF_BIBR].
However, neither the DUB inhibitor VLX1570 or EOAI nor the depletion of key DUBs, USP14, and UCH37 30, abolished the effect of TRIB2 in Bel-7402 and SMMC-7721 cells, excluding the possibility that TRIB2 regulates Ub via these DUBs.
Typically inhibiting USP14 promotes autophagy in M1-like macrophages and alleviates CLP-induced sepsis Macrophages , with diverse functions and variable phenotypes , are considered as an important executor of inflammatory diseases .
More specifically, in feedforward circuits G GABA --> Tgt does not limit the PSP peak (XREF_FIG), but increases in G GABA --> Tgt do limit the integration window (XREF_FIG) and the net total excitation (XREF_FIG).
This, in turn, represents a key mechanism by which these proteins influence enterocytic differentiation since the SLFN12 effects are blocked by pharmacologic inhibition or molecular reduction of the deubuiquitylases USP14 and UCHL5.
Whereas reducing USP14 activity can have beneficial effects in distinct disease models, overexpression of USP14 reduces mutant HTT aggregates and counteracts cell degeneration in neural cell lines expressing expanded-polyQ HTT constructs (Hyrskyluoto et al. 2014).
Perhaps in this active conformation, Usp14 and Ubp6 's C-terminus interacts with members of the ATPase ring to enable the C-termini of Rpt2 and/or Rpt5 to associate more tightly or for longer periods with the 20S 's intersubunit pockets.
Perhaps in this active conformation, Usp14 and Ubp6 's C-terminus interacts with members of the ATPase ring to enable the C-termini of Rpt2 and/or Rpt5 to associate more tightly or for longer periods with the 20S 's intersubunit pockets.
Interestingly, inhibition of USP14 alone by IU1 also reduced the phosphorylation levels of mTOR and p70S6K, but the phosphorylation levels of p38 MAPK was not altered.
Alternatively, USP14, a deubiquitnase that negatively regulates levels of RNF168 and suppresses RNF168 dependent ubiquitination signaling, is selectively recognized by p62 targeting for lysosomal degradation XREF_BIBR.
Thus Akt regulates radixin protein level not only through its T573 phosphorylation but also by regulating the rate of proteasomal degradation through cooperating with deubiquitinating protein, USP14 as precise control of UPS allows timely and selective degradation of radixin which is essential for its neural development.
The primers used were as follows : CD45 forward AAC AGT GGA GAA AGG ACG CA; CD45 reverse TGT GTC CAG AAA GGC AAA GC; and beta-actin primers as described earlier.
Here, we report that genetic and pharmacological inhibition of USP14 promotes mitophagy, which occurs in the absence of the well characterised mediators of mitophagy, PINK1 and Parkin.
Here, we report that genetic and pharmacological inhibition of USP14 promotes mitophagy, which occurs in the absence of the well characterised mediators of mitophagy, PINK1 and Parkin.
Interestingly, inhibition of USP14 alone by IU1 also reduced the phosphorylation levels of mTOR and p70S6K, but the phosphorylation levels of p38 MAPK was not altered.
Employing the overexpression of USP14 and the USP14 knockdown assay indicated that USP14 can greatly increase the steady-state levels of Acf7 by inhibiting the degradation of Acf7 through the ubiquitin- proteasome pathway.
Combined inhibition of USP7 and USP14 caused the degradation of HN1, as observed with inhibition of USP7 alone, but also destabilized DBN1, KHLC1, and SVIL, a known USP7 target (XREF_FIG, bottom).
However, inhibition of USP14 activity with IU1, silencing of UCH37 with siRNA, or a combination of both approaches to inhibit both USP14 and UCH37 simultaneously did not inhibit the release of IL-1beta (XREF_FIG).
Over-expression of USP14 reduces polyQ huntingtin aggregation and protects against cell degeneration by inhibiting IRE1alpha phosphorylation and blocking ER stress in PC6.3 cells.
Another recognized competitive DUB capable of inducing apoptosis in MM cells is VLX1570, which is a more potent and soluble analogue of b-AP15 able to strongly inhibit Ubiquitin carboxyl-terminal hydrolase 14 (UPS14) activity, inducing the accumulation of polyubiquitin chains, the expression of the chaperone HSP70B ', the oxidative stress marker heme oxygenase 1 (Hmox-1), and apoptosis in MM cells.
Additionally, USP14 inhibition increased the expression of Cx32 without changing its mRNA and ubiquitination levels, as showed by Real-time qPCR and immunoprecipitation assay respectively.
The underlined portion of the sequence (N-ALKITNECISCGACEPECPNEAISVG) was used to synthesize a probe composed of a 96-fold degenerate pool of oligomers having the composition [5 '-ATT (CA) ACN ATTIC) [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
We showed that LPS reduced the interaction between CBP and its degrader (FBXL19) as well as the abundance of FBXL19 and activated USP14, thus increasing CBP stability, enhancing histone acetylation, and promoting the expression of genes encoding proinflammatory cytokines.
Moreover, it has been revealed lately that the ubiquitin- specific protease 14 was instrumental in the catabolism of misfolded Htt by recruiting IRE1alpha and modulating the proteasome function, further adding to the complexity of HD pathology.[43]
Silencing of USP14 promoted proteasomal degradation of p-ERK (T202 and Y204) and p-Akt (T308 and S473), thus inactivating Akt and ERK signaling pathways.
Moreover, USP14 inactivation stimulated EIF2AK3/PERK- and ERN1/IRE1-mediated signaling pathways, which were responsible for VP16 degradation through SQSTM1/p62-mediated selective macroautophagy/autophagy.
Consistent with this, the de-ubiquitinase (DUB) USP14 appears to stimulate the signalling activity of Dvl by trimming its K63 linked ubiquitin chains [XREF_BIBR].
The Tgt enzyme accepts in addition to the E coil Q specific tRNAs allospecifically the corresponding tRNAs from other procaryotic and eukaryotic organisms.
Similarly, knockdown of UCH37 and USP14 or b-AP15 treatment rescued p53 protein, induced by COPS5 overexpression, and enhanced the activity of transfected luciferase reporter plasmids for p53, Bax, and p21 expression and protein levels of p53 downstream target genes BAX and p21.
Our experiments showed that CDK4, CDK6, CDK2, cyclinD1 and phosphorylated Rb were downregulated, while p27 and p15 were increased, by inhibiting USP14 expression or its activity in androgen responsive prostate cancer cells; and conversely, the exactly opposite changes were induced by USP14 overexpression.
Our experiments showed that CDK4, CDK6, CDK2, cyclinD1 and phosphorylated Rb were downregulated, while p27 and p15 were increased, by inhibiting USP14 expression or its activity in androgen responsive prostate cancer cells; and conversely, the exactly opposite changes were induced by USP14 overexpression.
Previous studies have shown that downregulation of USP14 leads to increased expression of E-cadherin and decreased expression of N-cadherin and vimentin in esophageal squamous cell carcinoma (ESCC) cells, as well as suppresses the migration and invasion of ESCC cells through the Wnt and beta-catenin signaling pathway.
Silencing of USP14 promoted proteasomal degradation of p-ERK (T202 and Y204) and p-Akt (T308 and S473), thus inactivating Akt and ERK signaling pathways.
We also found that inhibition of USP-14 and UCHL5 activities by the ITCs caused increased levels of USP14 and UCHL5 proteins, but not the third 19S deubiquitinating enzyme (DUB), POH1 and RPN11, suggesting feedback loop activation and further supporting that ITCs are inhibitors of proteasomal cysteine DUBs.
The human TRPV1 cDNA was then amplified with Pfu DNA polymerase (Fermentas) with specific forward and reverse primers, 5′-GAG GAT CCA GCA AGG ATG AAG AAA TGG AG-3′ and 5′-GAA TTC AAG GCC CAG TGT TGA C[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
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.
Specifically, upon the viral infections, TRIM14 could recruit USP14 to cut the lysine 48 (K48)-linked ubiquitin chains of cGAS at K414, causing inhibition of p62-mediated autophagic degradation of cGAS, therefore promoting the activation of type I interferon signaling to aid the elimination of the invading viruses (Chen et al., 2016).
SLFN12 or SERPB12 overexpression increases expression of the complementary deubiquitylases USP14 and UCHL5 in vitro, and SERPB12 stimulates USP14 deubiquitylase activity.
These results indicate that effect of S5 in Beclin1 polyubiquitination , Bcl2 and Beclin1 dissociation , autophagy activation , and M1 polarization inhibition is significantly dependent on USP14 .
RP ubiquitin receptors S5a and Rpn10 and Rpn13 capture substrates by recognizing their covalently attached ubiquitin chains, which are removed and disassembled by three deubiquitinating enzymes Rpn11, Ubp6 and Usp14 and Uch37 and UCHL5.
RP ubiquitin receptors S5a and Rpn10 and Rpn13 capture substrates by recognizing their covalently attached ubiquitin chains, which are removed and disassembled by three deubiquitinating enzymes Rpn11, Ubp6 and Usp14 and Uch37 and UCHL5.
Treatment of Akt inhibitor greatly increased radixin ubiquitination in the presence of USP14, indicating that Akt enhances protein stability of radixin not only through radixin phosphorylation but also through negatively regulates the UPS recruiting USP14 to radixin.
We next generated USP14 knockout (KO) cells using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology and found that the up-regulation of NF-kappaB activation in USP14 KO cells is totally reversed by NLRC5 siRNA.
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.
In this study, we identified and characterized an N-ethyl-N-nitrosourea (ENU) induced mutation in Usp14 (nmf375) that leads to adult-onset neurological disease.
There were no statistical differences in body weight among the three groups, but the size of xenograft tumors from USP14 knockdown HN6 cells were significantly smaller than those in the control group,[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Notably, the phosphorylation levels of USP14 were decreased in cells when treated with MK2206, an inhibitor of Akt (XREF_FIG), or when serum deprived, a condition known to inactivate endogenous Akt (XREF_FIG).
In addition, the OBs used in this research have shown their antiproliferative activity associated with their DUB-suppressing activities in a dose-dependent manner, suggesting that inhibition of UCHL5 and USP14 by OBs are functional, that would generate toxicity in normal tissues.
USP14 upregulation depends on NF-kappaB pathway activation, since inhibition of this pathway by ACHP, a selective inhibitor of IKK-beta, abolishes USP14 upregulation.
These results indicated that successful establishment of the rat model of AAC induced hypertrophy.To assess the potential involvement of USP14 in cardiac hypertrophy in vivo, Western blot analysis was[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The riboflavin mediated photosensitization reaction of a TGT trinucleotide with a lysine tripeptide (KKK) resulted in a major product [Gua-Lys (epsilon-amino)] that has a cross-link between the C8 of [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
The dicer siRNA substrates targeting the USP14 gene obtained from IDT (Coralville, Iowa), were encapsulated into lipid nanoparticles using microfluidic technology by Precision NanoSystems (SUB9KITS (TM); Vancouver, Canada).
Tgt stimulated crosslinking by the F937-736 CPR and inhibited crosslinking by U937-1139 CPR, an unfolded CPR involving a static partner to C937 (XREF_SUPPLEMENTARY).
CXCL12, a CXCR4 agonist, induces a time dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family.
Usp14 inhibition by 1D18 and 1B10 resulted in a slight decrease in retiree presentation while IU1 treatment did not diminish retiree presentation ( xref ).
In an angiotensin II (AngII) induced primary neonatal rat cardiomyocyte hypertrophy model, USP14 expression was increased in a time dependent manner, and reduced USP14 deubiquitinase activity or USP14 knockdown resulted in lower expression levels of the myocardial hypertrophy specific marker beta-MHC, and subsequent decreased GSK-3beta phosphorylation.
ART significantly increased TGT score, improved the survival of MNs, inhibited the oxidative stress, ameliorated the abnormal morphology of fibers in the musculocutaneous nerve, promoted the remyelination of axons, and alleviated muscle atrophy.
Notably, protein expression of the three key enzymes in the fatty acid synthesis pathway, ACLY, ACACA, FASN, and seven enzymes in the fatty acid degradation pathway were downregulated, which further indicated a possible role of USP14 in fatty acid metabolism.
Notably, protein expression of the three key enzymes in the fatty acid synthesis pathway, ACLY, ACACA, FASN, and seven enzymes in the fatty acid degradation pathway were downregulated, which further indicated a possible role of USP14 in fatty acid metabolism.
It was found that the administration of 6-Gingerol decreased the expression of USP14, greatly increased the number of autophagosomes, reactive oxygen species (ROS) and iron concentration, decreased the survival and proliferation rate of A549 cells, and significantly decreased tumor volume and weight.
To determine the interaction between AR and USP14 protein in the nucleus, we performed co-IP for AR and USP14 using both nuclear and cytosolic preparations of MDA-MB-453 cells treated with DHT or control solvent for 24h.
Analysis of the associated and non associated DUBs, such as UCHL2, USP14, UCH37, and UBC9 and DUB associated with core 20S proteasome (PSMD13) in RA-FLS showed that in the presence of IL-1beta, EGCG increased the expression of UCH37 and USP14, DUBs known to preferentially hydrolyze K48 linked polyubiquitin chains, with a marginal effect on unassociated DUB such as UCHL2 (XREF_FIG).