USP8 Data Analysis
- HGNC Gene Name
- ubiquitin specific peptidase 8
- HGNC Gene Symbol
- USP8
- Identifiers
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hgnc:12631
NCBIGene:9101
uniprot:P40818
- Orthologs
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mgi:1934029
rgd:1304979
- INDRA Statements
-
deubiquitinations
all statements
- Pathway Commons
-
Search for USP8
- Number of Papers
- 289 retrieved on 2023-02-19
DepMap Analysis
The Dependency Map (DepMap) is a genome-wide pooled
CRISPR-Cas9 knockout proliferation screen conducted in more than 700 cancer cell lines spanning many
different tumor lineages. Each cell line in the DepMap contains a unique barcode, and each gene
knockout is assigned a “dependency score” on a per cell-line basis which quantifies the rate of
CRISPR-Cas9 guide drop. It has been found that proteins with similar DepMap scores across cell
lines, a phenomenon known as co-dependent genes, have closely related biological functions. This can
include activity in the same or parallel pathways or membership in the same protein complex or the
same pathway.
We identified the strongest seven co-dependent genes (“Symbol”) for DUBs and ran GO enrichment
analysis. We used Biogrid, IntAct, and Pathway Commons PPIDs, and the NURSA protein-protein
interaction databases (PPIDs) to determine whether co-dependent genes interact with one another. The
“Evidence” column contains the PPIDs in which the interaction appears as well as whether there is
support for the association by an INDRA statement. As another approach to identify potential
interactors, we looked at proteomics data from the Broad Institute's Cancer Cell Line Encyclopedia (CCLE) for
proteins whose expression across ~375 cell lines strongly correlated with the abundance of each DUB;
it has previously been observed that proteins in the same complex are frequently significantly
co-expressed. The correlations and associated p-values in the CCLE proteomics dataset are provided.
And, we determined whether co-dependent genes yield similar transcriptomic signatures
in the Broad Institute's Connectivity
Map (CMap). A CMap score greater than 90 is considered significantly similar.
DepMap Correlations
Dependency GO Term Enrichment
Gene set enrichment analysis was done on the genes correlated with USP8using the
terms from Gene Ontology and gene sets derived from the
Gene Ontology Annotations database via
MSigDB.
Using the biological processes and other Gene Ontology terms from well characterized DUBs as a
positive control, several gene set enrichment analyses were considered. Threshold-less methods
like GSEA had relatively poor results.
Over-representation analysis with a threshold of of the top 7 highest absolute value Dependency Map
correlations yielded the best results and is reported below.
Transcriptomics
The following table shows the significantly differentially expressed genes after knocking
out USP8 using CRISPR-Cas9.
Knockout Differential Expression
Gene Set Enrichment Analysis
The GSEA method was applied for all genes whose knockout resulted in at least 20 significantly
differentially expressed genes.
Literature Mining
INDRA was used to automatically assemble known mechanisms
related to USP8 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
Deubiquitinase Activity
In addition, it has been reported that the deubiquitinase Usp8 could deubiquitinate Smo to influence Hh signaling activity.
Although our observations support the notion that USP8 deubiquitinates Smo and prevents localization to early endosomes, we are not suggesting that USP8 play an exclusive role in the inhibition of Smo endocytosis.
As shown in XREF_FIG, USP8, but not the other DUBs, reduced the ubiquitination of Myc-Smo.
Using an in vivo RNAi screen, we identified ubiquitin specific protease 8 (USP8) as a deubiquitinase that down-regulates Smo ubiquitination.
In addition, it has been reported that the deubiquitinase Usp8 could deubiquitinate Smo to influence Hh signaling activity (Li et al., 2012; Xia et al., 2012).
In addition, we provide evidence that the non visual beta-arrestin Krz acts in parallel with Smo ubiquitination to promote its internalization and that Smo ubiquitination is antagonized by the deubiquitinating enzyme UBPY.
However, we found that UBPY decreases Smo ubiquitination regardless of the Hh signaling states and that the association between UBPY and Smo is not significantly affected by either Hh stimulation or Smo phosphorylation, suggesting that Smo deubiquitination by UBPY is unlikely to be a major mechanism by which Hh inhibits Smo ubiquitination, although we can not rule out the possibility that Hh regulates UBPY binding to Smo in a subtle way that escaped the detection by our coimmunoprecipitation assay.
Taken together, these results suggest that UBPY can reverse Smo ubiquitination to promote its cell surface accumulation and induce low but not high levels of Hh pathway activation.
Hh promotes the formation of a Smo and USP8 complex, and USP8 further promotes the accumulation of Smo at the cell surface and prevents localization to the early endosomes by deubiquitinating Smo, leading to increased Hh signaling activity.
In an elegant and comprehensive analysis of corticotroph adenoma, Reincke et al demonstrated that heterozygous somatic USP8 single nucleotide mutation or deletions at or adjacent to the 14-3-3 protein binding domain make USP8 resistant to 14-3-3 protein binding and more prone to proteolytic cleavage, which, in turn, leads to higher rate of USP8 induced EGFR deubiquitination activity upon binding of EGF to its receptor.
USP8 (also known as UBPY) deubiquitylates EGFR on early endosomes, rescuing EGFR from degradation 107, 108 .
Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8.
However, we can not fully exclude the other possibility that the UBPY S680A expression resulted in a reduction in the cellular Ub conjugating activity toward activated EGFR in some way.The fact that U[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Based on these studies, we propose a model whereby the concerted recruitment of CHMP4B and UBPY to HD-PTP and the engagement of UBPY by STAM2 displaces ESCRT-0 from HD-PTP, deubiquitinates EGFR, and releases ESCRT-0 from cargo in favor of ESCRT-III.
Some studies showed that AMSH [31] and UBPY [32, 33] prevent EGFR down-regulation by deubiquitinating EGFR.
While AMSH is required for sorting of EGFR into MVEs and degradation in lysosomes XREF_BIBR, deubiquitination of EGFR by USP8 protects it from lysosomal degradation XREF_BIBR.
If USP8 deubiquitylates EGFR at the MVB, this facilitates EGFR 's progression toward degradation in the lysosome and, thus, aids receptor down-regulation.
Furthermore we demonstrated that both EGFR and EGFR-ErbB2 TM are deubiquitinated by the deubiquitination enzyme Usp8, although deubiquitination of ErbB2 was less efficient than that of EGFR [10].
Thus, deubiquitination of CHMP1B by USP8 at the endosomal membrane may favor CHMP1B oligomerization and co-assembly with IST1 in vivo.
Furthermore, we have demonstrated that USP8 deubiquitinates CHMP1B.
From these observations, we propose that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane associated ESCRT-III polymer.
We demonstrate further that CHMP1B is deubiquitinated by the ubiquitin specific protease USP8 (syn. UBPY)
Based on these observations, we propose that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane associated ESCRT-III polymer.
Our results thus strongly suggest that USP8 deubiquitinates CHMP1B.
Finally, we observed that the ubiquitination level of endogenous CHMP1B was higher in partially Usp8 silenced cells compared to control cells, strengthening the hypothesis that USP8 deubiquitinates CHMP1B (XREF_FIG).
Finally, deubiquitination of Parkin by USP8 is required for Parkin recruitment to CCCP intoxicated mitochondria and to promote stress induced mitophagy in vitro.
Our findings suggested that H 2 S promoted mitophagy formation by increasing S sulfhydration of USP8, which enhanced deubiquitination of parkin through the recruitment of parkin in mitochondria.
This process is negatively regulated by USP15 [XREF_BIBR] and USP30 [XREF_BIBR], which deubiquitinate mitochondrial Parkin-targets, while it is supported by USP8, which deubiquitinates Parkin itself [XREF_BIBR].
USP8 deubiquitylation of auto-ubiquitylated Parkin is required for its localization to depolarized mitochondria, and thereby for efficient activation of mitophagy [XREF_BIBR].
Whilst the phosphatase that dephosphorylates p-Ub remains unknown, two DUBs have been identified that deubiquitylate Parkin directed substrates, USP30 and USP15, and USP8 has also been reported to reverse Parkin autoubiquitylation.
USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin.
As USP8 interacts with CLK and expression of USP8-DN increases CLK ubiquitylation, the data indicate that USP8 deubiquitylates CLK, which down-regulates CLK and CYC transcriptional activity.
Since deubiquitylation of CLK by USP8 decreases its activity XREF_BIBR, it will be interesting to investigate whether CK2alpha phosphorylation affects CLK ubiquitylation.
CLK deubiquitylation by USP8 reinforces transcriptional repression by PER complexes, whereas CLK ubiquitylation and decreased phosphorylation may be involved in shifting CLK to a transcriptionally active state.
This rhythm in ubiquitylation is mediated by UBIQUITIN SPECIFIC PROTEASE 8 (USP8), which deubiquitylates CLK to downregulate CLK-CYC activity from ~ ZT18-ZT4, thereby reinforcing PER dependent repression [XREF_BIBR].
CLOCK deubiquitylation by USP8 inhibits CLK and CYC transcription in Drosophila.
USP8 directly deubiquitinates SQSTM1 and p62 and blocks autophagy [XREF_BIBR].
USP8 directly deubiquitinates SQSTM1 and p62 and blocks autophagy [XREF_BIBR].
USP8 induces the deubiquitination of TRAF6, TAB2, TAK1, p62, and BECN1, which are pivotal roles for NF-κB activation and autophagy induction.
USP8 overexpression leads to deubiquitination of p62 protein, suppressing its autophagic activity (Peng et al. 2020).
We provide evidence that Ubpy interacts with and deubiquitylates Hrs.
Mop recruits Ubpy to promote the deubiquitination of Hrs.
Studies have shown that USP8 also interacts with and deubiquitinate Hrs, demonstrating multiple roles of USP8 in both cargo de-ubiquitination and ESCRT-0 stability during development, which is helpful to address the mechanisms of Hh signaling.
Previous studies showed that Hrs is deubiquitinated by Ubpy.
Modified USP8 leads to the deubiquitination of SMO.
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Overexpression of Flag-USP8 in S2 cells reduced Smo ubiquitination (XREF_FIG, lane 3, top panel), whereas knockdown of USP8 by RNAi enhanced the levels of ubiquitinated Smo (XREF_FIG, lane 2, top panel), which was consistent with the data from the screen.
Consistent with UBPY being able to counteract Smo ubiquitination independent of Hh signaling states, overexpression of UBPY reduced Smo ubiquitination in S2 cells both in the absence and presence of Hh (XREF_FIG).
The overexpression of USP8 down-regulated Smo ubiquitination and increased Smo accumulation.
Moreover, overexpression of USP8 prevents Smo ubiquitination and elevates Smo accumulation, leading to increased Hh signaling activity.
USP8 modulates EGFR trafficking by regulating STAM de-ubiquitination on early endosomes 11.
In addition, we identified STAM and NFX1, which are known to be deubiquitylated by USP8 and USP9 respectively.
USP8 deubiquitinates STAM, preventing its degradation by the proteasome [XREF_BIBR], and Nrdp1, an E3 required for the lysosomal degradation of EGFR family members ErbB3 and ErbB4 [XREF_BIBR].
UBPY function is essential for effective downregulation but is likely to be multifaceted, encompassing activity against both K63-linked and K48-linked polyubiquitin chains and including regulation of the stability of ESCRT-associated proteins such as STAM, by reversing their ubiquitination.
Further, overexpression of wild-type USP8 accelerates channel deubiquitylation, while either a catalytically inactive mutant USP8 or siRNA mediated knockdown of USP8 enhanced accumulation of ubiquitylated KCa3.1, thereby inhibiting channel degradation.
Further, we demonstrated that KCa3.1 is initially ubiquitylated following endocytosis and then deubiquitylated by USP8 prior to lysosomal degradation XREF_BIBR.
USP8 reduces both multiple monoubiquitination and polyubiquitination of Cx43 to prevent autophagy mediated degradation.
The ubiquitin-specific protease USP8 deubiquitinates and stabilizes Cx43
USP8 interacts with and deubiquitinates Cx43, removing monoubiquitin moieties as well as K63- and K48 linked ubiquitin chains.
USP8 deubiquitinates EGFR phosphorylated on Y1172, Y1110, K867, K737, K754, K929, Y1092, Y1016, Y1197, K970, and Y1069 on K716.
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USP8 depleted endothelial cells displayed altered VEGFR2 ubiquitination and production of a unique VEGFR2 extracellular domain proteolytic fragment caused by VEGFR2 accumulation in the endosome-lysosome system.
We now provide evidence that USP8 de-ubiquitinates VEGFR2.
Conversely, the de-ubiquitinating enzyme, USP8, is shown to mediate de-ubiquitination of VEGFR2, regulating VEGFR2 trafficking, proteolysis, and signal transduction 39.
USP8 deubiquitinates EGFR phosphorylated on Y1172, Y1110, K737, K754, K929, Y1092, Y1016, K716, Y1197, K970, and Y1069 on K867.
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USP8 deubiquitinates EGFR phosphorylated on Y1172, Y1110, K867, K737, K754, K929, Y1092, Y1016, K716, Y1197, and Y1069 on K970.
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ERBB2 is a target for USP8-mediated deubiquitination
We recently showed that Usp8 also deubiquitinates ERBB2, albeit to a much lesser extent than EGFR [17].
We recently showed that Usp8 also deubiquitinates ErbB2, albeit to a much lesser extent than EGFR [10].
We concluded that USP8 deubiquitinates Sec31A and inhibits the formation of large COPII carriers, thereby suppressing collagen IV secretion.
Ubiquitin-specific protease 8 deubiquitinates Sec31A and decreases large COPII carriers and collagen IV secretion
Here, we show that the deubiquitinating enzyme USP8 interacts with and deubiquitinates Sec31A.
USP8 deubiquitinates EGFR phosphorylated on Y1172, Y1110, K867, K754, K929, Y1092, Y1016, K716, Y1197, K970, and Y1069 on K737.
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To determine whether endogenous USP8 deubiquitinates LRIG1, we used shUSP8 to decrease the level of endogenous USP8 in EBC1 cells.
Also, when over-expressed, USP8 decreases LRIG1 ubiquitination by SAIT301 treatment (XREF_FIG).
TrkB deubiquitination by USP8 regulates receptor levels and BDNF dependent neuronal differentiation.
TrkB deubiquitination by USP8 regulates receptor levels and BDNF-dependent neuronal differentiation.
TrkB deubiquitination by USP8 regulates receptor levels and BDNF dependent neuronal differentiation.
USP8 deubiquitinates EGFR phosphorylated on Y1172, Y1110, K867, K737, K929, Y1092, Y1016, K716, Y1197, K970, and Y1069 on K754.
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We found that the carboxy terminal domain of Nrdp1 binds to the rhodanese domain of USP8, and that USP8 very efficiently deubiquitinates and stabilizes Nrdp1.
USP8 deubiquitinates STAM, preventing its degradation by the proteasome [XREF_BIBR], and Nrdp1, an E3 required for the lysosomal degradation of EGFR family members ErbB3 and ErbB4 [XREF_BIBR].
USP8 deubiquitinates EGFR phosphorylated on Y1172, Y1110, K867, K737, K754, Y1092, Y1016, K716, Y1197, K970, and Y1069 on K929.
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USP8 Deubiquitinates SHANK3 to Control Synapse Density and SHANK3 Activity-Dependent Protein Levels
USP8 acts to deubiquitinate SHANK3, which prevents its proteasomal mediated degradation and enhances overall dendritic spine stability.
USP8 Deubiquitinates SHANK3 to Control Synapse Density and SHANK3 Activity Dependent Protein Levels.
USP8 and USP9X deubiquitinate ITCH to induce ubiquitination and degradation of the anti-apoptotic protein c-FLIP, leading to apoptosis in glioblastoma [XREF_BIBR], or to anoikis in pancreatic ductal adenocarcinoma [XREF_BIBR].
Expression of the catalytically inactive mutants, AMSH(D348A) and UBPY(C786S), caused an increase in PAR(2) ubiquitination and trapped the receptor in early endosomes, thereby preventing lysosomal trafficking and degradation.
USP8 and AMSH mediate deubiquitination of PAR2 and its sorting from endosomes to lysosomes [XREF_BIBR].
Furthermore, shRNA mediated knockdown of USP8 is sufficient to enhance the basal level of AMPAR ubiquitination in primary neurons.
In addition to USP46, USP8 can also deubiquitinate mammalian AMPARs indicating that multiple regulatory mechanisms exist to control AMPAR ubiquitination levels (Scudder et al., 2014).
HIF1alpha deubiquitination by USP8 is essential for ciliogenesis in normoxia.
Bland and colleagues suggested that leptin increases the expression of USP8, which in turn deubiquitylates the leptin receptor by cleaving Lys48-ubiquitin chains, among other (still unknown) chain types.
USP8 deubiquitinates the leptin receptor and is necessary for leptin mediated synapse formation.
We propose that deubiquitination of EPG5 by USP8 guards the autophagic flux in ESCs to maintain their stemness.
Mechanistically, USP8 directly removes non-classical K63-linked ubiquitin chains from EPG5 at Lysine 252, leading to enhanced interaction between EPG5 and LC3.
In addition, this study identifies USP8 as one of the best markers of Lewy bodies in human pigmented neurons in sporadic cases of Parkinson’s disease and demonstrates the ability of USP8 to hydrolyze K63-linked ubiquitin chains from α-synuclein in vitro
Another deubiquitinase, USP8, removes K63-linked ubiquitin chains of α-synuclein and prevents its lysosomal degradation.
The identified USP8 mutants increase EGFR deubiquitination to inhibit EGF induced EGFR downregulation, leading to augmented and more sustained EGFR signaling.
USP8 mutants diminished epidermal growth factor receptor ubiquitination and induced Pomc promoter activity in immortalized AtT-20 corticotropinoma cells.
Consistent with this idea, overexpression of wild-type USP8 decreased the ubiquitination of the FLIP (S) E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIP (S) interaction, whereas siRNA mediated suppression of USP8 increased AIP4 ubiquitination.
Consistent with this idea, over-expression of WT USP8 decreased ubiquitination of the FLIP S E3 ubiquitin ligase AIP4, an event previously shown to increase AIP4-FLIP S interaction, while siRNA mediated suppression of USP8 increased AIP4 ubiquitination.
Finally, even though CYT-1 shows ligand induced K63 polyubiquitination, it is not subjected to deubiquitination by the K63 polyubiquitin specific AMSH deubiquitinating enzyme, while CYT-1 is slightly deubiquitinated by USP8.
Finally, CYT-1 is not subjected to deubiquitination by the K63 polyubiquitin specific AMSH DUB enzyme, while CYT-1 is slightly deubiquitinated by USP8.
These data further demonstrate that the two isoforms of Otubain 1 have opposing effects on GRAIL and that Otubain 1 ARF-1 recruits the ubiquitin specific protease 8 (USP-8) to promote GRAIL deubiquitination and stabilization.
This unexpected function of otubain-1 might be mediated through the inhibition of USP8, a DUB that binds to and deubiquitylates GRAIL; however, it is not known how otubain-1 might inhibit USP8.
USP8 directly deubiquitylates and stabilizes FLIPL
USP8 and USP9X deubiquitinate ITCH to induce ubiquitination and degradation of the anti-apoptotic protein c-FLIP, leading to apoptosis in glioblastoma [XREF_BIBR], or to anoikis in pancreatic ductal adenocarcinoma [XREF_BIBR].
UBPY also deubiquitinated Eps15 in vitro, suggesting that Eps15 is a cellular substrate for UBPY.
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We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway.
We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway.
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Synaptic Strength Is Bidirectionally Controlled by Opposing Activity-Dependent Regulation of Nedd4-1 and USP8
Indeed, the Y1045F and Y1091F Cbl binding site mutants are deubiquitinated by Usp8, suggesting that this ubiquitination signal may represent mono- or oligo-ubiquitin (Figs. 7 and 8, upper panel, arrow[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
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The Cbl binding site mutants of EGFR (Y1045F) and EGFR-ErbB2 (Y1091F) are also deubiquitinated by Usp8 both with and without EGF stimulation (Figs. 7 and 8).
Smoothened, a key component of Hedgehog pathway, is deubiquitinated by USP8 34 and activation of Hedgehog pathway induces ACTH secretion 35.
USP8 induces the deubiquitination of TRAF6, TAB2, TAK1, p62, and BECN1, which are pivotal roles for NF-κB activation and autophagy induction.
The Cbl binding site mutants of EGFR (Y1045F) and EGFR-ErbB2 (Y1091F) are also deubiquitinated by Usp8 both with and without EGF stimulation (Figs. 7 and 8).
Furthermore we demonstrated that both EGFR and EGFR-ErbB2 TM are deubiquitinated by the deubiquitination enzyme Usp8, although deubiquitination of ErbB2 was less efficient than that of EGFR [10].
However, we can not fully exclude the other possibility that the UBPY S680A expression resulted in a reduction in the cellular Ub conjugating activity toward activated EGFR in some way.The fact that U[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
In contrast, Fzd receptors are deubiquitinated by UBPY and ubiquitin specific protease 6 and 8 (USP6 and USP8).
USP8 regulates another EGFR family member, ErbB3 by modulating Nrdp1 (neuregulin-receptor-degradation protein-1)
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Recent studies have attempted to identify the specific DUBs associated with TrkA, where Ceriani and collaborators described an interaction between TrkA and USP8 (USP-family) in PC12 cells
Our data are consistent with the recent findings that USP8 directly deubiquitylates and stabilizes the long isoform of FLICE like inhibitory protein (FLIP L) in cervical cancer cell line ME-180, which was derived from the metastatic site of epidermoid carcinoma.
For example, in this and a previous study we observed that neither USP34, nor USP8 deubiquitinate CFTR, and only USP10 activity was inhibited by Cif XREF_BIBR, XREF_BIBR.
In addition, we identified STAM and NFX1, which are known to be deubiquitylated by USP8 and USP9 respectively.
BRUCE regulates DNA double-strand break response by promoting USP8 deubiquitination of BRIT1
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USP8 induces the deubiquitination of TRAF6, TAB2, TAK1, p62, and BECN1, which are pivotal roles for NF-κB activation and autophagy induction.
USP8 interacted with ENaC, as detected by co-immunoprecipitation, and it deubiquitinated ENaC.
USP8 in the endosome deubiquitinates EGFR in the endosome.
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We conclude that UBPY negatively regulates the rate of EGFR down-regulation by deubiquitinating EGFR on endosomes.
Conversely, deubiquitination of TCHP is mediated by ubiquitin-specific peptidase 8 (USP8) [50].
The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation
SAIT301 treatment significantly enhanced the ubiquitination of LRIG1, whereas over-expression of USP8 markedly diminished the ubiquitination of LRIG1 (XREF_FIG).
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In addition to USP46, USP8 can also deubiquitinate mammalian AMPARs indicating that multiple regulatory mechanisms exist to control AMPAR ubiquitination levels (Scudder et al., 2014).
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Indeed, the Y1045F and Y1091F Cbl binding site mutants are deubiquitinated by Usp8, suggesting that this ubiquitination signal may represent mono- or oligo-ubiquitin (Figs. 7 and 8, upper panel, arrow[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
USP8 directly deubiquitylates and stabilizes FLIP L, but not the short isoform.
Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import impaired mutant.
These data suggest a reciprocal E3-DUB relationship in which GRAIL can ubiquitinate USP8, and ubiquitinated USP8 can de-ubiquitinate GRAIL.
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In D. melanogaster models, the ubiquitin conjugating enzyme UBE2E3 promotes ubiquitination of TDP-43; in contrast, ubiquitin isopeptidase Y (UBPY) decreased TDP-43 ubiquitination.
Other Statements
USP8 depletion accelerates receptor turnover, whereas loss of hepatocyte growth factor regulated substrate (Hrs) rescues this phenotype, indicating that USP8 protects EGFR from degradation via an Hrs dependent pathway.
Previous work reported that USP8 depletion severely inhibits EGFR degradation 11.
Other studies have suggested that mutations in USP8 reduce the degradation of EGFR, such as HER-2 and HER-3, thereby promoting tumor progression.
Finally, depletion of endogenous UBPY by RNA interference resulted in elevated ubiquitination and accelerated degradation of EGF activated EGFR.
Nevertheless, both AMSH [31, 34, 35] and UBPY [32, 33, 36-38] have been reported to increase EGFR down-regulation.
We propose a model in which the coordinated action of UBE4B, ESCRT-0, and the deubiquitinating enzyme USP8 enable the endosomal sorting and lysosomal degradation of the EGFR.
In other studies, both AMSH XREF_BIBR, XREF_BIBR and UBPY XREF_BIBR, XREF_BIBR, XREF_BIBR have been reported to increase EGFR down-regulation.
USP8 knockdown leads to reduce EGFR protein and inhibits ACTH secretion.
Whereas USP8 variants increase EGFR signaling in cultured cells, such an effect has not been consistently shown in vivo, suggesting that the effect is small or temporary or that other factors [XREF_BIBR] are involved in increased ACTH production and/or proliferation of USP8 mutation positive tumors.
Depletion of UBPY, another DUB that associates with ESCRT components, has been shown to both accelerate and slow the rate of EGFR degradation.
We found that USP8 mutated PAs have a higher incidence of EGFR expression, increased EGFR protein abundance and activation of downstream Erk1/2, indicating that USP8 mutations enhance EGFR signaling in tumors.
In human corticotroph primary cultures, treatment with the pan-ErbB TKI canertinib as well as the EGFR TKI gefitinib suppresses POMC mRNA, and USP8 mutations, detected in up to two-thirds of CD, may underlie the increase in EGFR signaling in these tumors.
USP8 mutations, detected in up to two-thirds of Cushing disease, may underlie the increase in EGFR signalling in these tumours.
The USP8 mutation (14-3-3 somatic mutations) inhibits EGFR degradation, enhances EGFR accumulation, and consequently, likely induces corticotroph EGFR tumor signaling.
Considering that activation of EGFR-MAPK signaling induces p27 (Kip1) degradation, and p27 (Kip1)-deficient mice develop corticotropinoma XREF_BIBR, XREF_BIBR, we speculate that through activating EGFR signaling USP8 mutation accelerates p27 (Kip1) degradation, representing an important molecular mechanism underlying ACTH hyperproduction (XREF_FIG).
In keeping with this intermediate phenotype, UBPY depletion partially reduced the interaction of EGFR with ESCRT-III.
While one report suggests that Usp8 inhibits EGFR degradation [25], we and others have demonstrated that Usp8 promotes EGFR degradation [21,26].
By the same mechanism, USP8 also directs the trafficking and lysosomal degradation of CXCR4 [XREF_BIBR], MET and epidermal growth factor receptor [XREF_BIBR, XREF_BIBR], implying that its loss consequent to increased RNF41 abundance would prolong and potentially amplify invasion signaling by these receptors.
By removing the lysosomal sorting signal, UBPY negatively regulates the downregulation of EGFR [10].
While some reports suggest that Usp8 inhibits EGFR degradation [40], we and others demonstrated that Usp8 stimulates EGFR degradation [41,42].
USP8 has been shown to have opposing effects on EGF receptor degradation by either directly deubiquitinating receptors to prevent their degradation, or by deubiquitinating ESCRT complex proteins to stabilize them and thus promote EGF receptor degradation [XREF_BIBR, XREF_BIBR, XREF_BIBR - XREF_BIBR].
Depletion of USP8 with siRNA inhibits EGFR activation and increases EGFR degradation [32], similar to the effect of depleting SEPW1, and suggests the possibility SEPW1 might regulate USP8.
Consistently, the expression of EGFR was decreased by genetic silencing of USP8.
Western blotting confirmed that knockdown of USP8 not only reduced RTK phosphorylation, but also the total levels of EGFR, ERBB2, ERBB3, and MET in H1975 and H1650 cells (XREF_FIG).
Taken together, our findings demonstrate for the first time that inhibition of USP8 down-regulates the total protein levels of EGFR, ERBB2, ERBB3, and MET, and effectively attenuates related RTK signaling pathways.
USP8 knockdown in primary ACTH secreting tumor cells, however, reduced ACTH secretion and EGFR levels, suggesting that inhibition of USP8 activity may be an effective treatment strategy for CD.
Moreover, USP8 knockdown reduced EGFR protein level in USP8 mutated tumor cells (XREF_SUPPLEMENTARY).
Modified USP8-C748A increases the amount of ubiquitinated EGFR.
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We previously showed that overexpression of the Usp8 C748A mutant strongly enhances accumulation of the steady state level of ubiquitinated EGFR in the absence of EGF [41].
Importantly, USP8 inactivation attenuated ACTH secretion and EGFR expression in primary tumor cells.
Overexpression of UBPY reduced the ubiquitination level of EGFR and delayed its degradation in EGF stimulated cells.
The stimulation of Hh promotes Smo deubiquitination by ubiquitin specific protease 8 (USP8), which blocks Smo endocytosis and enhances Smo cell surface accumulation XREF_BIBR XREF_BIBR.
Overexpression of USP8 caused an elevation of Smo in both A- and P-compartment cells but did not ectopically activate Hh target genes, such as ptc, in A-compartment cells located away from the A/P boundary (XREF_FIG).
In addition, a gain-of-function experiment showed that the overexpression of USP8 caused an accumulation of Smo in both A- and P-compartment cells (XREF_FIG) and caused anterior expansion of ptc-lacZ in cells that received Hh (XREF_FIG).
We next wished to assess whether the accumulation of Smo induced by the overexpression of USP8 or the inactivation of Shi was due to changes in the cell surface accumulation of Smo.
We revealed that overexpression of usp8 blocked rack1 RNAi induced Smo degradation, suggesting that Rack1 positively regulates Smo possibly through Usp8.
These data suggest that USP8 promotes the cell surface accumulation of Smo, and that Shi mediates Smo endocytosis.
USP8 Promotes Smo Signaling Activity.
USP8 Promotes Smo Accumulation in Wing Imaginal Discs.
It is also possible that USP8 promotes Smo recycling to the cell surface.
The overexpression of USP8 down-regulated Smo ubiquitination and increased Smo accumulation.
Moreover, overexpression of USP8 prevents Smo ubiquitination and elevates Smo accumulation, leading to increased Hh signaling activity.
USP8 Prevents the Localization of Smo to the Early Endosome.
The stimulation of Hh promotes Smo deubiquitination by ubiquitin specific protease 8 (USP8), which blocks Smo endocytosis and enhances Smo cell surface accumulation XREF_BIBR XREF_BIBR.
Moreover, we found that USP8 prevents Smo localization to early endosomes that are labeled with Rab5.
USP8 increases the amount of SMO.
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We also provide evidence that the non visual beta-arrestin Kurtz (Krz) acts in parallel with Smo ubiquitination to control Smo cell surface expression, and that the deubiquitinating enzyme UBPY promotes Smo cell surface expression by counteracting Smo ubiquitination.
Our explanation is that the increase in Smo levels that was induced by USP8 was still inhibited by Ptc, since we found that USP8 induced more severe overgrowth phenotypes in the ptc mutant background (XREF_FIG).
UBPY may modulate Smo cell surface expression by attenuating Smo endocytosis and/or promoting Smo recycling (XREF_FIG).
However, although the overexpression of USP8 increased the levels of Smo in vivo, it failed to induce Hh target gene expression in A-compartment cells located away from the A/P boundary.
Overexpression of USP8 elevated the level of Smo but did not induce ectopic Hh signaling activity in A compartment cells located away from the A/P boundary.
USP8 decreases the amount of SMO.
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Overexpression of Flag-USP8 in S2 cells reduced Smo ubiquitination (XREF_FIG, lane 3, top panel), whereas knockdown of USP8 by RNAi enhanced the levels of ubiquitinated Smo (XREF_FIG, lane 2, top panel), which was consistent with the data from the screen.
Modified USP8 decreases the amount of ubiquitinated SMO.
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In agreement with this hypothesis, the overexpression of HA-USP8 reversed the effects of Flag-USP8C> S and again reduced the levels of ubiquitinated Smo (XREF_FIG, lane 4, top panel).
The overexpression of USP8 decreased the amount of Smo that co-localized with Rab5 (XREF_FIG), which suggested that USP8 may prevent the accumulation of Smo in early endosomes and therefore promote the cell surface accumulation of the protein (XREF_FIG) as well as increase the signaling activity (XREF_FIG).
We also observed that down-regulation of USP8 inhibited the proliferation of GC cells which highly expressed HER-3.
Indeed, inhibition of USP8 either by its knockdown or synthetic small molecule led to attenuation of variety of receptor tyrosine kinase (RTK) activities, resulting in the inhibition of cell proliferation in gefitinib-resistant and -sensitive non-small cell lung cancer (NSCLC) cells [47] .
USP8 Inhibitor Suppresses HER-2 Positive Gastric Cancer Cell Proliferation and Metastasis via the PI3K and AKT Signaling Pathway.
In conclusion, USP8 inhibitor could inhibit proliferation, abolishes clonogenic ability, and induces apoptosis in pituitary corticotroph tumor cell-AtT20 cells.
Down-regulation of USP8 Inhibits Cholangiocarcinoma Cell Proliferation and Invasion.
Erratum: Down-Regulation of USP8 Suppresses HER-3 Positive Gastric Cancer Cells Proliferation [Corrigendum].
Both GADD45beta and CABLES1 may be responsible, at least in part, for the USP8 induced suppression of corticotroph tumor cell proliferation.
Therefore, it was confirmed that down-regulation of USP8 could inhibit the proliferation of NCI-N87, MKN-45 and AGS cell lines, which is HER-3 positive GC cells.
All in vitro results demonstrated that down-regulation of USP8 inhibited the proliferation and viability of GC cells with high expression of HER3 (NCI-N87, MKN-45 and AGS), but did not affect HER3 negative cells (MGC-803).
Down-Regulation of USP8 Suppresses HER-3 Positive Gastric Cancer Cells Proliferation.
We demonstrated that knockdown of USP8 significantly inhibited the proliferation, migration and invasion of QBC939 and RBE cells in vitro, while USP8 overexpression showed significant promoting effects on Hucct-1 cells.
Indeed, inhibition of USP8 either by its knockdown or synthetic small molecule led to attenuation of variety of receptor tyrosine kinase (RTK) activities, resulting in the inhibition of cell proliferation in gefitinib resistant and -sensitive non small cell lung cancer (NSCLC) cells [XREF_BIBR].
However, although overexpressing USP8 in SiHa and SW756 cells enhanced cell proliferation, it did not reach statistical significance according to our data.
KD of the EGF receptor (EGFR) in human RPE cells inhibits USP8 Tyr717 and Tyr810 phosphorylation, which enables TCHP and AURKA degradation and reverses the serum-induced effects on ciliogenesis and cell proliferation [50].
We will provide an overview of corticotroph tumorigenesis in the context of hypothalamic-pituitary-adrenal (HPA) axis regulation with an emphasis on the role of the glucocorticoid receptor in the resistance to the negative feedback of cortisol that occurs in CD, and we will explore the role of epidermal growth factor receptor (EGFR) signaling in ACTH hyper-secretion and corticotroph cell proliferation and the recent discovery of somatic ubiquitin specific peptidase 8 (USP8) mutations in a significant number of patients with sporadic CD with an emphasis on therapeutic implications.
CCK-8 cell viability tests showed that USP8 can promote tumor cell proliferation, although statistical significance was not achieved (XREF_FIG).
Cellular studies showed that USP8 can enhance the proliferation, migration, and invasion abilities of CSCC cells, thereby promoting tumor progression.
Furthermore, by overexpressing or silencing USP8, cellular studies indicated that USP8 can directly upregulate the proliferation and metastatic abilities of CSCC cells.
Moreover, silencing of USP8 also promoted apoptosis in cholangiocarcinoma cells by regulating the Bcl-2 and Bax axis and Caspase cascade; up-regulation of USP8 decreased apoptosis in Hucct-1 cells.
High expression of USP8 can therefore inhibit extrinsic apoptosis by stabilizing FLIP L [ xref ] .
Consistently, knockdown of USP8 significantly increased apoptosis of PIK3CA mutant cells even in the presence of glutamine (XREF_SUPPLEMENTARY).
In the present study , our objective is to study in broad the secondary down-stream effect after depleting USP5 or USP8 , which were initially showed to induce apoptosis in various cancers .
Taken together, our data indicate that USP8 functions as a novel deubiquitylase of FLIP L and inhibits extrinsic apoptosis by stabilizing FLIP L.
USP8 DUB prevents c-FLIP L, degradation and further halts the apoptosis in cancer cells suggesting it to be a potential drug target.
High expression of USP8 can therefore inhibit extrinsic apoptosis by stabilizing FLIP L [XREF_BIBR].
USP8 inhibition via genetic and pharmacological approaches resulted in growth inhibition and apoptosis induction in both sensitive and doxorubicin resistant HCC cells.
Knockdown of USP8 inhibited the proliferation , migration , invasion , and cell cycle progression of A549 and H1299 cells , and promoted the apoptosis .
Knockdown of USP8 inhibited the proliferation of human lung cancer cells by regulating cell cycle- and apoptosis related proteins.
The results indicated that the down-regulation of USP8 could significantly promote the apoptosis of NCI-N87 and MKN-45 cells, but it did not work on MGC-803 cells (XREF_FIG).
These results indicated that down-regulation of USP8 could promote the apoptosis of HER3 positive GC cells and inhibit the proliferation of them by affecting the cell-cycle.
Knockdown of USP8 inhibited the proliferation, migration, invasion, and cell cycle progression of A549 and H1299 cells, and promoted the apoptosis.
In the present study, our objective is to study in broad the secondary down-stream effect after depleting USP5 or USP8, which were initially showed to induce apoptosis in various cancers.
Moreover, down-regulation of USP8 could promote the apoptosis of HER3 positive GC cells and inhibit the proliferation of them by affecting the cell-cycle.
USP8 knockdown leads to reduce EGFR protein and inhibits ACTH secretion.
USP8 mutated tumors are more common in females, and are associated with earlier onset, a smaller size, and increased ACTH production.
For the first time, we showed that USP8 knockdown or gefitinib treatment significantly reduced ACTH secretion in primary USP8 mutated corticotrophin adenoma cells, but not in wild-type cells.
We further showed that USP8 knockdown or gefitinib (a clinically available EGFR inhibitor) treatment significantly reduced ACTH secretion in primary USP8 mutated corticotrophin adenoma cells, but not in wild-type cells.
Because EGFR signaling increases POMC transcription and secretion of ACTH [XREF_BIBR], increased USP8 activity causes elevated ACTH production.
The presence of such associations is supported by the finding that USP8 knockdown or EGFR inhibition attenuates ACTH secretion in primary USP8 mutated tumor cells [XREF_BIBR].
We will provide an overview of corticotroph tumorigenesis in the context of hypothalamic-pituitary-adrenal (HPA) axis regulation with an emphasis on the role of the glucocorticoid receptor in the resistance to the negative feedback of cortisol that occurs in CD, and we will explore the role of epidermal growth factor receptor (EGFR) signaling in ACTH hyper-secretion and corticotroph cell proliferation and the recent discovery of somatic ubiquitin specific peptidase 8 (USP8) mutations in a significant number of patients with sporadic CD with an emphasis on therapeutic implications.
USP8 knockdown using shRNA in primary corticotroph adenoma cells effectively reduced ACTH production.
It is expected that USP8 mutations deregulate these molecules to drive ACTH production and secretion.
While USP8 mutations were less likely to enhance tumorous ACTH hypersecretion via EGFR mediated activation, the presence of USP8 mutations may predict favorable responses to the somatostatin analog pasireotide, which exhibits high affinity for SSTR5.
It is expected that USP8 mutations deregulate these molecules to drive ACTH production and secretion.
Ultimately, USP8 mutants lead to inhibition of EGF signaling downregulation and increased Pomc expression and ACTH secretion [XREF_BIBR, XREF_BIBR].
Down-Regulation of USP8 Promotes the Degradation of HER-3.
Erratum: Down-Regulation of USP8 Suppresses HER-3 Positive Gastric Cancer Cells Proliferation [Corrigendum].
All these results indicated that down-regulation of USP8 could inhibit the proliferation of HER-3 positive cells, NCI-N87 and MKN-45, in vivo.
These results are opposite of the results seen by Niendorf et al. [130] showing that deletion of USP8 leads to lower level of ERBB3.
Western blotting confirmed that knockdown of USP8 not only reduced RTK phosphorylation, but also the total levels of EGFR, ERBB2, ERBB3, and MET in H1975 and H1650 cells (XREF_FIG).
Down-regulation of USP8 inhibited cell proliferation and cell metastasis and also reduced the HER-3 expression.
USP8 or USP9X silencing increased HER3 protein level in basal conditions (medium alone) in BxPC3 cells, suggesting that each deubiquitinase promotes basal HER3 degradation by stabilizing ITCH, as proposed for USP8 [XREF_BIBR].
These results indicated that down-regulation of USP8 could promote the apoptosis of HER3 positive GC cells and inhibit the proliferation of them by affecting the cell-cycle.
Moreover, down-regulation of USP8 could promote the apoptosis of HER3 positive GC cells and inhibit the proliferation of them by affecting the cell-cycle.
In contrast, USP8 knockdown suppressed melanoma cell growth, survival and migration, and augmented the inhibitory effects of therapeutic drugs.
USP8 was originally identified to enhance cell growth as its expression increases upon serum stimulation in cancer cells.
USP8 is known to enhance cell growth as its expression increases in cancer cell XREF_BIBR.
Small molecule inhibitors of USP8 (HBX90,397 and HBX 90,659) have been shown to inhibit HCT116 and PC3 cell growth, and to display specificity for USP8 among a panel of cysteine proteases [XREF_BIBR].
XREF_BIBR Ubiquitin specific peptidases (USP8) was originally identified to enhance cell growth as its expression increases upon serum stimulation in cancer cells.
Since Nrdp1 targets USP8 for ubiquitylation, we speculated that overexpression of Nrdp1 could enhance protein ubiquitylation and USP8 degradation in PC12 cells.
USP8 markedly enhanced the stability of Nrdp1, and a point mutant that disrupts USP8 catalytic activity destabilized endogenous Nrdp1.
Our results indicate that Nrdp1 is a specific target for the USP8 deubiquitinating enzyme and are consistent with a model where USP8 augments Nrdp1 activity by mediating its stabilization.
Moreover, we conducted cellular studies and found that USP8 can significantly upregulate the migration and invasion processes of CSCC cell lines.
USP8 promotes CSCC progression by enhancing tumor invasion capacity.
However, both the migration and invasion capacities of CSCC cells were upregulated by USP8 overexpression (XREF_FIG).
In conclusion, we demonstrated that USP8 in cancer tissue is an independent prognostic biomarker of CSCC, and high USP8 in CSCC can enhance cell invasion.
We demonstrated that knockdown of USP8 significantly inhibited the proliferation, migration and invasion of QBC939 and RBE cells in vitro, while USP8 overexpression showed significant promoting effects on Hucct-1 cells.
Cellular studies showed that USP8 can enhance the proliferation, migration, and invasion abilities of CSCC cells, thereby promoting tumor progression.
Down-regulation of USP8 Inhibits Cholangiocarcinoma Cell Proliferation and Invasion.
Knockdown of USP8 inhibited the proliferation, migration, invasion, and cell cycle progression of A549 and H1299 cells, and promoted the apoptosis.
EGFR activates USP8 by phosphorylating Tyr 717 and Tyr 810.
EGFR activation causes USP8 to become phosphorylated and to associate with EGFR on endosomes where it dynamically regulates EGFR ubiquitination state during trafficking.
Most importantly, epidermal growth factor receptor (EGFR) kinase activated USP8 by phosphorylating Tyr 717 and Tyr 810.
Importantly, GST-EGFR phosphorylated non-tagged USP8 and elevated its DUB activity toward ubiquitin oligomers (Fig. xref ; lanes 1–3), but GST-EGFR did not activate GST-USP8 (Supplementary Fig. xref ).
Taken together, EGFR activates USP8 by directly phosphorylating Tyr 717 and -810 in vitro.
EGFR activates USP8 by phosphorylating Tyr-717 and Tyr-810.
Taken together, EGFR activates USP8 by directly phosphorylating Tyr-717 and −810 in vitro.
Considering that activation of EGFR-MAPK signaling induces p27 (Kip1) degradation, and p27 (Kip1)-deficient mice develop corticotropinoma XREF_BIBR, XREF_BIBR, we speculate that through activating EGFR signaling USP8 mutation accelerates p27 (Kip1) degradation, representing an important molecular mechanism underlying ACTH hyperproduction (XREF_FIG).
USP8 enhances mitophagy by removing lysine-6-linked ubiquitin from parkin, promoting its turnover [XREF_BIBR].
And USP8 catalyzes ubiquitin removal from both Lys48 linkage and Lys63 linkage polyubiquitination chains.
71 In contrast to PAR2, USP8 (or AMSH) does not impact CXCR4 ubiquitination but instead modulates the ubiquitin status of ESCRT-0 that is ubiquitinated by the E3 ligase AIP4, 23 reinforcing the idea that ubiquitination of the transport machinery represents an important regulatory event in GPCR trafficking.
Moreover, P5091 inhibits USP7-, but not USP2- or USP8 mediated cleavage of poly K48 linked ubiquitin chains (visualized by the presence or absence of mono-ubiquitin) (XREF_SUPPLEMENTARY).
This zinc-finger ribbon (observed also in USP2, USP8 and USP21 structures) is in the contracted ' closed-hand ' configuration seen in USP8, which was proposed to block ubiquitin access XREF_BIBR.
In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species.
This zinc-finger ribbon seems to be in the contracted " closed-hand " configuration seen in USP8 that blocks ubiquitin access (XREF_FIG).
Perturbed VEGFR2 endosomal trafficking caused by USP8 depletion could modulate endosome linked signal transduction.
VEGFR2 accumulation also occurred when cells were treated with individual USP8 siRNAs to limit off-target effects.
VEGFR2 also accumulated in EEA1 positive early endosomes when cells were treated with individual USP8 siRNAs to limit off-target effects.
VEGF-A-stimulated VEGFR2 signal transduction is perturbed by USP8 depletion.
USP8 decreases the amount of KDR.
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Accumulation of proteolytic cleavage products in USP8 depleted cells reduced levels of mature VEGFR2 and resulted in lower levels of ubiquitinated full-length receptor.
When Nrdp1 is increased, more USP8 will be degraded by Nrdp1, and as a return, less USP8 will make Nrdp1 unstable, resulting in less Nrdp1 and more USP8 in the cells.
RNF41 also negatively regulates the stability of the ubiquitin isopeptidase USP8, a regulator of JAK2 associated cytokine receptor sorting and processing : elevated RNF41 destabilizes the endosomal-sorting-complexes-required-for-transport (ESCRT) -0 complex (Hrs and signal-transducing-and adapter-molecule (STAM) -1 or STAM2), which results in cargo arriving at sorting endosomes being routed to recycling endosomes rather than to lysosomes [XREF_BIBR].
Since Nrdp1 targets USP8 for ubiquitylation, we speculated that overexpression of Nrdp1 could enhance protein ubiquitylation and USP8 degradation in PC12 cells.
Nrdp1 activates NRG-1beta-induced HER3 degradation [XREF_BIBR] and USP8 prevents Nrdp1 auto-ubiquitination [XREF_BIBR], with a strong correlation between USP8 expression and Nrdp1 stabilization [XREF_BIBR].
Since Nrdp1 targets USP8 for ubiquitylation, we speculated that overexpression of Nrdp1 could enhance protein ubiquitylation and USP8 degradation in PC12 cells.
RNF41 redistributes and ubiquitylates USP8, and reduces USP8 levels.
USP8 KD also prevented Parkin KO DA neurons loss and normalized mitochondrial morphological defects, although it did not ameliorate Parkin climbing performance (XREF_FIG).
Only USP8 supports mitophagy by stabilizing the E3 ligase Parkin.
H 2 S may upregulate the expression of deubiquitinating enzymes USP8 to antagonize the degradation of Parkin protein.
In contrast, USP8 promotes parkin mediated mitophagy and thus agonists of this DUB could be developed XREF_BIBR.
In contrast, USP8 promotes Parkin mediated mitophagy and agonists to USP8 could be developed as potential therapeutics.
Overexpression of USP8 increased c-FLIP S ubiquitination, decreased FLIP S half-life, decreased FLIP S steady-state levels, and decreased TRAIL resistance (XREF_FIG).
Over-expression of WT USP8, but not catalytically inactive USP8, increased FLIP S ubiquitination, decreased FLIP S half-life, decreased FLIP S steady-state levels, and decreased TRAIL resistance, while siRNA mediated suppression of USP8 levels had the opposite effects.
Furthermore, the suppression of FLIP S levels by USP8 over-expression was reversed by introduction of siRNA targeting AIP4.
Loss of PTEN function (right panel, XREF_FIG), in contrast increases pAkt levels, decreases USP8 levels, and turns off the USP8 and AIP4 ubiquitin switch, allowing FLIP S to accumulate and suppress TRAIL induced apoptosis.
Because PTEN dependent regulation of FLIP S stability is mediated by changes in FLIP S ubiquitination, we took the above USP8 modulated cells, transiently transfected a construct encoding HA tagged ubiquitin, and following FLIP S immunoprecipitation used Western blot analysis to monitor the effect of USP8 alteration on the extent of HA-ubiquitin incorporated into FLIP S.
Taken together, our data indicate that USP8 functions as a novel deubiquitylase of FLIP L and inhibits extrinsic apoptosis by stabilizing FLIP L.
Collectively, these data demonstrate that the Cbl binding site of the wild-type EGFR and the EGFR-ErbB2 chimera is not required for efficient EGF induced Usp8 tyrosine phosphorylation or coprecipitati[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
EGF treatment induced a faster degradation of EGFR protein in HeLa cells expressing WT USP8 compared to mutants, although EGFR protein levels were comparable in these cells under serum starved conditions (XREF_FIG).
As we have demonstrated before, deletion of the MIT-domain in Usp8 results in decreased EGF induced Usp8 tyrosine phosphorylation [17].
pS722 was only found in EGF stimulated cells containing Usp8 C748A, which could indicate that this serine is involved in substrate trapping.
In EGFR-ErbB2 expressing cells, EGF induced Usp8 tyrosine phosphorylation was even lower in the presence of PD153035 than the Usp8 tyrosine phosphorylation level observed in unstimulated and mock trea[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
In cells expressing either EGFR or EGFR-ErbB2, removal of the MIT domain (Usp8 Delta140) resulted in a decrease of the EGF induced Usp8 tyrosine phosphorylation, when compared to Usp8 wt.
Another hypothesis is that USP8 is catalytically inhibited in a phosphorylation dependent manner by 14-3-3 proteins during the interphase stage of the cell-cycle, and this regulation is reversed in the M phase [XREF_BIBR].
Another hypothesis is that USP8 is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3 proteins during the interphase stage of the cell-cycle, and this regulation is reversed in the M phase [ xref ].
We conclude that UBPY is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3s during the interphase, and this regulation is cancelled in the M phase.
Mechanistically, we show that Hh promotes the interaction of USP8 with Smo aa625-753, which covers the three PKA and CK1 phosphorylation clusters.
CFP-Smo was transfected into S2 cells that were treated with GFP dsRNA (control), USP8 dsRNA, or Shi dsRNA and then treated with Hh conditioned medium or control medium.
We show here that UBPY silencing also activates autophagy in absence of CCCP treatment, which strongly suggests that UBPY is not connected exclusively to mitophagy.
Inactivation of human UBPY in HeLa cells activates autophagy.
Lastly, we have shown that shRNA mediated inactivation of UBPY in HeLa cells also affects autophagy which appears to be deregulated with an increased number of autophagosomes and increased autophagy flux.
We show here that UBPY silencing also activates autophagy in absence of CCCP treatment, which strongly suggests that UBPY is not connected exclusively to mitophagy.
In addition, COS-7 cells have fair amount of endogenous USP8, which perhaps have already increased hOAT1 expression and activity to certain extent.
The protein levels of cell membrane protein marker E-cadherin (XREF_FIG, bottom panel) and cell total protein marker beta-actin (XREF_FIG, bottom panel) were not affected under these conditions, thereby indicating that the change in hOAT1 expression induced by USP8 wild type transfection was not due to the general perturbation of membrane and cellular proteins.
In Fig. 10 , USP8 slowed down the rate of hOAT1 degradation as compared to that in control cells ( empty vector-transfected cells ) , suggesting that USP8 played a role in increasing hOAT1 stability .
Downexpression of USP8 decreased OAT1 transport activity and OAT1 expression.
In XREF_FIG, USP8 slowed down the rate of hOAT1 degradation as compared to that in control cells (empty vector transfected cells), suggesting that USP8 played a role in increasing hOAT1 stability.
In the present study the linkage between PTEN loss, Akt activation, and USP8 levels and activity appeared to be somewhat different, and in our work Akt activation decreased, rather than enhanced, USP8 function by increasing USP8 ubiquitination and decreasing steady-state USP8 levels (data not shown).
AKT increases the amount of USP8.
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The AKT kinase has been previously shown to decrease USP8 function by increasing USP8 ubiquitination and decreasing active steady-state USP8 level [XREF_BIBR].
The AKT kinase has been previously shown to decrease USP8 function by increasing USP8 ubiquitination and decreasing active steady-state USP8 level [XREF_BIBR].
In addition to suppressing levels of USP8, Akt may also stimulate the activity of USP8 toward select targets such as Nrdp1.
These results suggest that simultaneous blockage of USP8 may further enhance LRIG1 dependent Met degradation and subsequent tumor growth inhibition by SAIT301 and other Met targeting drugs that have a similar mechanism of action.
Over-expression of USP8 substantially reduced the LRIG1 degradation (XREF_FIG).
Moreover, we showed that suppression of USP8 activity, by over-expression of USP8-CS, led to enhancement of the anti-tumor activity of Met targeting therapeutic antibody by promoting degradation of both Met and LRIG1.
This result suggests that SAIT301 perturbs USP8 mediated modulation of LRIG1, resulting in the degradation of LRIG1.
Loss of USP8/UBPY blocked Hh-induced Smo accumulation whereas overexpression of USP8/UBPY resulted in ectopic Smo accumulation and Hh pathway activation [42,43].
Although the loss-of-function of USP8 can block Hh induced cell surface accumulation of Smo, we found that USP8 only stabilizes Smo but does not regulate Smo phosphorylation in the absence of Hh (XREF_FIG), suggesting that the regulation of Smo by USP8 is downstream of Smo phosphorylation.
Moreover, we found that USP8 prevents Smo localization to early endosomes that are labeled with Rab5.
USP8 Prevents the Localization of Smo to the Early Endosome.
Moreover, Shi RNAi attenuated the localization of Smo in the early endosomes (XREF_FIG), whereas USP8 RNAi elevated the localization (XREF_FIG).
Catalytic inactivation of USP8 incurs EGFR hyperubiquitination and promotes receptor localization to endosomes marked by high ubiquitin content.
siRNAs USP8 (B) and (C) reduced expression of MET and STAM2.
UBPy deficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2.
Hence, locally recruited UBPY might aid the displacement of STAM2 from HD-PTP.
These binding reactions provide a scenario in which UBPY could aid transit of EGFR to ESCRT-III by helping to displace STAM2 from HD-PTP.
USP8 activity thus modulates VEGF-A-stimulated Akt and ERK1/2 activation but does not affect other VEGFR2 associated signal transduction pathways.
Importantly, knockdown of USP8 inhibited activation of the Akt signaling pathway by decreasing the phosphorylation level of Akt and up-regulated p53 expression, while USP8 overexpression increased activation of the Akt signaling pathway in Hucct-1 cells.
Whereas ERK1/2 and Akt signaling is inhibited by USP8 depletion, other signal transduction pathways are unaffected.
USP8 decreases the amount of AKT.
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Importantly, knockdown of USP8 inhibited activation of the Akt signaling pathway by decreasing the phosphorylation level of Akt and up-regulated p53 expression, while USP8 overexpression increased activation of the Akt signaling pathway in Hucct-1 cells.
USP8 overexpression blocked NGF induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8 and UBPy (C748A) caused a marked increase of cell differentiation.
USP8 overexpression blocked NGF induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8 and UBPy (C748A) caused a marked increase of cell differentiation.
The deubiquitinating enzyme UBPy and USP8 interacts with TrkA and inhibits neuronal differentiation in PC12 cells.
Here, we provide mechanistic evidence indicating that ubiquitin carboxyl-terminal hydrolase 8 (USP8) modulates BDNF and TrkB dependent neuronal differentiation.
Over-expression of WT USP8, but not catalytically inactive USP8, increased FLIP S ubiquitination, decreased FLIP S half-life, decreased FLIP S steady-state levels, and decreased TRAIL resistance, while siRNA mediated suppression of USP8 levels had the opposite effects.
Overexpression of USP8 increased c-FLIP S ubiquitination, decreased FLIP S half-life, decreased FLIP S steady-state levels, and decreased TRAIL resistance (XREF_FIG).
Overexpression of USP8 increased c-FLIP S ubiquitination, decreased c-FLIP S half-life, decreased c-FLIP S steady-state levels, and decreased TRAIL resistance.
USP8 increased ENaC current in Xenopus oocytes and collecting duct epithelia and enhanced ENaC abundance at the cell surface in HEK 293 cells.
Thus, USP8 and USP2-45 selectively modulate ENaC trafficking at different steps in the endocytic pathway.
This resulted from altered endocytic sorting; USP8 abolished ENaC degradation in the endocytic pathway, but it had no effect on ENaC endocytosis.
This unexpected function of otubain-1 might be mediated through the inhibition of USP8, a DUB that binds to and deubiquitylates GRAIL; however, it is not known how otubain-1 might inhibit USP8 (Ref. xref ).
This unexpected function of otubain-1 might be mediated through the inhibition of USP8, a DUB that binds to and deubiquitylates GRAIL; however, it is not known how otubain-1 might inhibit USP8.
Interestingly, Otub1 expression completely abolished USP8 mediated stabilization of GRAIL when all 3 proteins were co-expressed.
These data suggest that USP8 phosphorylation, possibly on Tyr residue (s), enhances its DUB activity.
The putative substrate of BRUCE could be USP8 and if this is the case, ubiquitination of USP8 by BRUCE is expected to enhance its Dub activity over Ub-BRIT1, thereby facilitating removal of the Ub chains from K63-Ub-BRIT1 and promoting recruitment of de-ubiquitinated BRIT1 to sites of DNA damage.
Furthermore, the catalytic activity of USP8 is inhibited by phosphorylation on Ser 680, based on the fact that the S680A mutant of USP8 exhibites enhanced DUB activity toward polyubiquitin chains and EGFR.
Altogether , RNAi for UBR4 , HGS , STAM , and USP8 led to the upregulation of 672 proteins and to the downregulation of 797 proteins , compared to a control RNAi ( white RNAi ) .
Overall , there were 15 proteins that were similarly upregulated by RNAi for UBR4 , STAM , HGS , and USP8 , compared to a control RNAi .
Altogether , RNAi for UBR4 , HGS , STAM , and USP8 led to the upregulation of 672 proteins and to the downregulation of 797 proteins , compared to a control RNAi ( white RNAi ) .
Interestingly, the inhibition of USP8 suppresses growth of gefitinib resistant non small cell lung cancer cells, though no link to the potential impact on HIF-1alpha is reported.
Silencing or pharmacological inhibition of USP8 deubiquitinase, relevant in particular to the stability of RTKs such as EGFR and MET, was shown to induce death of gefitinib resistant NSCLC cells in vitro and in vivo [XREF_BIBR].
Knock-down of USP8 selectively decreases viability of gefitinib resistant NSCLC cells.
USP8 down-regulation completely prevented the loss of PINK1 KO DA neurons (XREF_FIG), restoring dopamine to wild-type levels (XREF_FIG).
USP8 KD also prevented Parkin KO DA neurons loss and normalized mitochondrial morphological defects, although it did not ameliorate Parkin climbing performance (XREF_FIG).
Heterozygous USP8 gene deletion (USP8 -/+) in PINK1 KO background also completely prevented the loss of DA neurons (XREF_FIG), restored dopamine levels to wild-type (XREF_FIG), corrected thoracic muscle fiber disorganization (XREF_FIG) and mitochondrial structure (XREF_FIG), ameliorated the shorter longevity (XREF_FIG), and completely corrected the locomotor defects (XREF_FIG).
KD of the EGF receptor (EGFR) in human RPE cells inhibits USP8 Tyr717 and Tyr810 phosphorylation, which enables TCHP and AURKA degradation and reverses the serum-induced effects on ciliogenesis and cell proliferation [50].
In zebrafish, knockout (KO) of kctd17 impairs ciliogenesis in Kupffer’s vesicle and induces situs inversus [74], whereas KO of usp8 increases ciliogenesis in the pronephric duct and causes renal cysts [50].
CRL3KCTD17 , USP8 , and TCHP TCHP , a centriolar protein originally identified as a keratin-binding protein , activates AURKA and suppresses ciliogenesis [ 101,141,142 ] .
USP8 down-regulation completely prevented the loss of PINK1 KO DA neurons (XREF_FIG), restoring dopamine to wild-type levels (XREF_FIG).
USP8 down-regulation rescues mitochondria defects of PINK1 KO flies.
Both USP8 fly lines (USP8 +/- and USP8 RNAi) restored complex I activity of PINK1 mutants (XREF_FIG).
USP8 decreases the amount of MET.
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Ubiquitinated LRIG1 recruits c-Met to lysosomes for degradation; reduction in LRIG1 ubiquitination by USP8 depletion or inactivation thus enables c-Met levels and functionality to be maintained.
USP8 increases the amount of MET.
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We further found that USP8 inhibition decreased levels of multiple receptor tyrosine kinases (RTKs) by ~ 90%, such as epidermal growth factor receptor (EGFR) and c-Met.
The USP8 inhibited HER-2 positive GC cell proliferation and migration in vivo and in vitro and probably served as a novel potential therapeutic biomarker for HER-2 positive GC.
USP8 Inhibitor Suppresses HER-2 Positive Gastric Cancer Cell Proliferation and Metastasis via the PI3K and AKT Signaling Pathway.
Western blotting confirmed that knockdown of USP8 not only reduced RTK phosphorylation, but also the total levels of EGFR, ERBB2, ERBB3, and MET in H1975 and H1650 cells (XREF_FIG).
Inhibiting USP8 leads to decrease in IL-7ra mRNA as well as Ccr7 [81].
Inhibiting USP8 leads to decrease in IL-7ra mRNA as well as Ccr7 [ 81 ] .
Inhibiting USP8 leads to decrease in IL-7ra mRNA as well as Ccr7 [XREF_BIBR].
KD of the EGF receptor (EGFR) in human RPE cells inhibits USP8 Tyr717 and Tyr810 phosphorylation, which enables TCHP and AURKA degradation and reverses the serum-induced effects on ciliogenesis and cell proliferation [50].
CRL3KCTD17 , USP8 , and TCHP TCHP , a centriolar protein originally identified as a keratin-binding protein , activates AURKA and suppresses ciliogenesis [ 101,141,142 ] .
KD of the EGF receptor (EGFR) in human RPE cells inhibits USP8 Tyr717 and Tyr810 phosphorylation, which enables TCHP and AURKA degradation and reverses the serum-induced effects on ciliogenesis and cell proliferation [50].
Taken together, our findings suggested that luteolin inhibits microglial inflammation by enhancing USP8 protein production.
Western blot analysis verified that USP8 expression is upregulated by luteolin.
We next hypothesized that luteolin inhibits microglial inflammation by regulating USP8 gene expression.
Moreover, silencing of USP8 also promoted apoptosis in cholangiocarcinoma cells by regulating the Bcl-2 and Bax axis and Caspase cascade; up-regulation of USP8 decreased apoptosis in Hucct-1 cells.
USP8 increases the amount of BAX.
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The results of western blot indicated that knockdown of USP8 down-regulated the expression of Cyclin D1, CDK4, CDK6, p-AKT, and Bcl2, and up-regulated the expression of Bax.
USP8 decreases the amount of BAX.
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The results of western blot indicated that knockdown of USP8 down-regulated the expression of Cyclin D1, CDK4, CDK6, p-AKT, and Bcl2, and up-regulated the expression of Bax.
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USP8 promotes Parkin-mediated mitophagy by deubiquitinating Parkin and promoting its recruitment to the mitochondria .
Usp8 knockdown impairs Parkin-mediated mitophagy by preventing Parkin recruitment of depolarized mitochondria .
USP8 overexpression also reduced the production of lipopolysaccharide (LPS)-induced proinflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2).
USP8 overexpression also reduced the production of lipopolysaccharide (LPS)-induced proinflammatory mediators such as inducible nitric oxide synthase (iNOS), nitric oxide (NO), cyclooxygenase-2 (COX-2), and prostaglandin E2 (PGE2).
The present study is the first, to the best of our knowledge, to investigate the effects of ubiquitin specific peptidase 8 (USP8) on IHR induced inflammation in renal tubular epithelial cells and examine the underlying mechanism.
Effect of deubiquitinase USP8 on hypoxia and reoxygenation induced inflammation by deubiquitination of TAK1 in renal tubular epithelial cells.
Additionally, the deubiquitinating enzymes UBPY and USP6 may promote the recycling of endocytosed FZD back to the surface and restore Wnt signaling.
USP8 and UBPY is reported to activate the Wnt and beta-catenin pathway by targeting Frizzled G protein coupled protein XREF_BIBR.
In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species.
Furthermore, knockdown of UBPY promotes formation of insoluble TDP-43 aggregates and induced neurotoxicity in D. melanogaster.
USP8 Inhibitor Suppresses HER-2 Positive Gastric Cancer Cell Proliferation and Metastasis via the PI3K and AKT Signaling Pathway.
XREF_BIBR, XREF_BIBR Therefore, it can be inferred that down-regulation of USP8 may inhibit the proliferation and even metastasis of GC through this pathway.
Inhibiting USP8 leads to decrease in IL-7ra mRNA as well as Ccr7 [XREF_BIBR].
Inhibiting USP8 leads to decrease in IL-7ra mRNA as well as Ccr7 [81].
Interestingly, HAT1 is upregulated also by USP8 loss but it is not regulated by RNAi for HGS, STAM.
Interestingly , HAT1 is upregulated also by USP8 loss but it is not regulated by RNAi for HGS , STAM .
Another DUB enzyme, USP8, functions similarly to target Eps15 [XREF_BIBR].
Furthermore, inactivation of UBPY caused the accumulation of Eps15 on the endosomal aggregates.
Cleavage of USP8 led to increased deubiqutination of the EGF receptor, impairing its downregulation and sustaining EGF signaling.
XREF_BIBR Cleavage of USP8 led to increased deubiquitination of the epidermal growth factor receptor (EGFR), impairing its downregulation and sustaining EGF signaling.
Silencing or pharmacological inhibition of USP8 deubiquitinase, relevant in particular to the stability of RTKs such as EGFR and MET, was shown to induce death of gefitinib resistant NSCLC cells in vitro and in vivo [XREF_BIBR].
USP8 siRNAs reduced viability and increased death in cSCC lines but had little effect in normal skin cells (XREF_FIG a).
USP8 inhibition markedly reduced cell viability in gefitinib resistant and -sensitive NSCLC cells, but exhibited no observable effects on normal control cells (XREF_FIG).
H1975 and H1650 transfected with si-USP8 showed a dramatic decrease in cell viability compared to mock transfected cells, indicating that the suppression of USP8 effectively decreases NSCLC cell viability (XREF_FIG and XREF_SUPPLEMENTARY).
Indeed, inhibition of USP8 either by its knockdown or synthetic small molecule led to attenuation of variety of receptor tyrosine kinase (RTK) activities, resulting in the inhibition of cell proliferation in gefitinib-resistant and -sensitive non-small cell lung cancer (NSCLC) cells [47] .
Studies have shown that RNAi mediated depletion of USP8 increased BACE1 ubiquitination on Lys 501, promoted BACE1 accumulation in the early endosomes and late endosomes and lysosomes, and decreased levels of BACE1 in the recycling endosomes.
Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes and lysosomes, and decreased levels of BACE1 in the recycling endosomes.
Therefore, we found that down-regulation of USP8 could significantly inhibit the cell-cycle of AGS and block the G1 phase.
Therefore, it was confirmed that down-regulation of USP8 could inhibit the proliferation of NCI-N87, MKN-45 and AGS cell lines, which is HER-3 positive GC cells.
Furthermore, the finding that CHMP1B is a target of USP8 may shed new light in the future on understanding its contribution to membrane receptor trafficking, resistance to chemotherapy or EGFR stabilization in Cushing 's disease.
CHMP1B is a target of USP8 and UBPY regulated by ubiquitin during endocytosis.
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These data indicate that under ischemic conditions, Nrdp1 upregulation may hinder the stabilization of HIF-1alpha in neurons via promoting ubiquitin mediated degradation of USP8, thus attenuating cellular adaptive response to hypoxia and ischemia.
Nbr1 binds additionally to proteins implicated in ubiquitin mediated protein turnover and vesicle trafficking : ubiquitin specific peptidases USP8, and the endosomal transport regulator p14 and Robld3.
VEGF-A-stimulated VEGFR2 signal transduction is perturbed by USP8 depletion.
The inhibition of USP8 downregulated the Notch signalling pathway via NICD destabilization, resulting in the retardation of cellular growth, wound closure, and colony forming ability of breast cancer cell lines.
The catalytic domain of USP8 (USP8NT3) was sufficient to reduce Smo ubiquitination (XREF_FIG, lane 4, top panel), enhance the accumulation of endogenous Smo (XREF_FIG), and increase the GFP-Smo-mediated induction of ectopic dpp-lacZ and ptc expression (XREF_FIG, XREF_SUPPLEMENTARY '' ').
Co-expression of Flag-USP8 with GFP-Smo by MS1096 Gal4 caused induction of dpp-lacZ and ptc expression in A-compartment cells both close to and away from the A/P boundary (XREF_FIG), whereas the expression of Flag-USP8 alone did not induce ectopic dpp-lacZ and ptc expression (XREF_FIG).
Knockdown of USP8 inhibited the proliferation, migration, invasion, and cell cycle progression of A549 and H1299 cells, and promoted the apoptosis.
These data clearly indicate that USP8 depletion induces the loss of trichoplein and causes the ciliogenesis dependent cell cycle arrest.
KD of the EGF receptor (EGFR) in human RPE cells inhibits USP8 Tyr717 and Tyr810 phosphorylation, which enables TCHP and AURKA degradation and reverses the serum-induced effects on ciliogenesis and cell proliferation [50].
KD of the EGF receptor (EGFR) in human RPE cells inhibits USP8 Tyr717 and Tyr810 phosphorylation, which enables TCHP and AURKA degradation and reverses the serum-induced effects on ciliogenesis and cell proliferation [50].
USP8 increases the amount of NRG.
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Mechanistically, we found that USP8 increased the expression of neuregulin receptor degradation protein-1 (Nrdp1), potently downregulated the expression of TLR4 and myeloid differentiation primary response protein 88 (MyD88) protein, and inhibited the phosphorylation of IkappaB kinase (IKK) beta and kappa B-alpha (IkappaBalpha), thereby reducing nuclear translocation of p65 by inhibiting the activation of the nuclear factor-kappaB (NF-kappaB) signaling pathway in LPS induced mice.
Researchers have found that USP8 markedly enhanced the stability of neuregulin receptor degradation protein-1 (Nrdp1), which in turn inhibited the production of proinflammatory cytokines in toll like receptor triggered macrophages.
This de-ubiquitination activity has made USP8 a stabilizing molecule for HIF-1alpha protein, in which USP8 prevents HIF-1alpha from pVHL mediated degradation.
These data suggest that Nrdp1 may attenuate neuron 's adaptive response to hypoxia and ischemia via interfering USP8 mediated HIF-1alpha stabilization, thus contributing to neuronal death under ischemic conditions.
UBPy deficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2.
Similarly, the overexpression of Ubpy can also rescue the loss of Hrs in mop knockdown by removing the ubiquitin moiety from Hrs.
Whereas ERK1/2 and Akt signaling is inhibited by USP8 depletion, other signal transduction pathways are unaffected.
USP8 activity thus modulates VEGF-A-stimulated Akt and ERK1/2 activation but does not affect other VEGFR2 associated signal transduction pathways.
In Drosophila, USP8 was recently reported to decrease CLK activity by deubiquitylation XREF_BIBR.
CLOCK deubiquitylation by USP8 inhibits CLK and CYC transcription in Drosophila.
Moreover, USP8 protein regulates the differentiation and proper BDNF dependent dendritic formation of hippocampal neurons in vitro and in vivo We conclude that USP8 positively regulates the levels and activation of TrkB modulating BDNF dependent neuronal differentiation.
Here, we provide mechanistic evidence indicating that ubiquitin carboxyl-terminal hydrolase 8 (USP8) modulates BDNF and TrkB dependent neuronal differentiation.
Loss of PTEN function (right panel, XREF_FIG), in contrast increases pAkt levels, decreases USP8 levels, and turns off the USP8 and AIP4 ubiquitin switch, allowing FLIP S to accumulate and suppress TRAIL induced apoptosis.
Conversely, exposure of PTEN mutant human GBM cells to an Akt inhibitor enhanced USP8 levels (last lane, XREF_FIG).
LEP increases the amount of USP8.
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Bland and colleagues suggested that leptin increases the expression of USP8, which in turn deubiquitylates the leptin receptor by cleaving Lys48-ubiquitin chains, among other (still unknown) chain types.
Bland and colleagues suggested that leptin increases the expression of USP8 , which in turn deubiquitylates the leptin receptor by cleaving Lys48-ubiquitin chains , among other ( still unknown ) chain types .
Quantification of immunoblot data showed that whereas ligand stimulated VEGFR2 ubiquitination displayed a characteristic peak and decline, under conditions of USP8 depletion VEGFR2 ubiquitination persisted.
Quantification of VEGFR2 residing in the endosome-lysosome system upon CHX treatment revealed VEGFR2 levels were 30% higher in non stimulated, USP8 depleted cells.
The fused GST might perturb the regulatory mechanism of USP8.
Importantly, GST-EGFR phosphorylated non-tagged USP8 and elevated its DUB activity toward ubiquitin oligomers (Fig. xref ; lanes 1–3), but GST-EGFR did not activate GST-USP8 (Supplementary Fig. xref ).
Addition of GST-14-3-3epsilon dose-dependently inhibited the activity of wild-type UBPY as judged by the increased and decreased levels of Ub chains (especially Ub 4-7) and Ub monomer (Ub 1), respecti[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
By contrast, GST-14-3-3epsilon did not inhibit the activity of UBPY S680A which does not interact with 14-3-3s.
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Dithiothreitol, a reducing agent that reverses sulfhydration mediated covalent modification, increased the ubiquitylation level of parkin, abolished the effects of exogenous H 2 S on USP8 deubiquitylation and suppressed the interaction of USP8 with parkin in neonatal rat cardiomyocytes treated with high glucose, oleate and palmitate.
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HeLa cells transfected with FLAG-UBPY were treated with or without nocodazole, and FLAG-UBPY was immunopurified from their lysates in the same way as in Fig. 3.
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In addition to diminishing RNF41 auto-ubiquitination, which results in increased RNF41 expression, lenalidomide treatment increased USP8 expression and basal expression and signaling of EpoR and interleukin-3 receptors, without altering expression of Type II receptors such as c-Kit and IFNAR.
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RA-9, a chalcone derivative with a structure similar to b-AP15, was reported to inhibit proteasomal DUBs [XREF_BIBR] as well as UCHL1, UCHL3, USP2, USP5, and USP8 [XREF_BIBR].
Our group found that NMDAR activation negatively regulates AMPAR ubiquitination, suggesting that the influx of calcium through NMDAR channels activates USP8.
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In keeping with our previous results we found an increased number of total autophagy vesicles in UBPY inactivated cells or in control cells treated with bafilomycin A1 (XREF_SUPPLEMENTARY).
HBX 90,397, another DUB-specific inhibitor, blocks USP8 activity.
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Ectopic expression of USP8 mutants or cleaved USP8 in murine corticotroph cell line induced higher POMC promoter activity and transcription than wild-type USP8.
The effect was specific for USP8 because re-expression of USP8 in USP8 RNAi cells restored MFN levels (XREF_FIG).
Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1.
Inhibition of the DUBs AMSH and UBPY inhibit the early-to-late endosomal sorting of protease activated receptor 2 (PAR2) [XREF_BIBR].
Moreover, USP8 downregulated several pro inflammatory cytokines [nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), prostaglandin E 2 (PGE 2), and interleukin-1beta (IL-1beta)] in the serum and brain, and the relevant protein factors [inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2)] in the brain.
Recurrent gain-of-function mutations in USP8 cluster at the 14-3-3 binding site and impair the binding of 14-3-3 proteins.
Moreover, USP8 downregulated several pro inflammatory cytokines [nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), prostaglandin E 2 (PGE 2), and interleukin-1beta (IL-1beta)] in the serum and brain, and the relevant protein factors [inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2)] in the brain.
We also found that USP8 siRNA blocked luteolin inhibition of pro inflammatory gene expression such as iNOS, NO, COX-2, and PGE2.
Targeting USP8 can reduce the expression of growth factor receptors that participate in cSCC development.
These binding reactions provide a scenario in which UBPY could aid transit of EGFR to ESCRT-III by helping to displace STAM2 from HD-PTP.
Taken together, our data indicate that USP8 functions as a novel deubiquitylase of FLIPL and inhibits extrinsic apoptosis by stabilizing FLIPL.
USP8 suppresses death receptor-mediated apoptosis by enhancing FLIPL stability.
Moreover, inactivation of USP8 by either USP8RNAi or USP8C> S in Drosophila embryo down-regulated En expression (XREF_FIG, compare to wild-type En staining in H) and caused lethality of the embryo or pupa (unpublished data).
The stimulation of Hh promotes Smo deubiquitination by ubiquitin specific protease 8 (USP8), which blocks Smo endocytosis and enhances Smo cell surface accumulation XREF_BIBR XREF_BIBR.
While the enzymes mediating SHANK ubiquitination are not known , it has been recently found that USP8 selectively deubiquitinates SHANK1 and SHANK3 and modulates dendritic spine density and morphology ( Campbell and Sheng , 2018 ) .
In this study, we therefore tried to address the question whether human and canine CD are caused by the same hotspot mutations in the USP8 gene.
Ectopic expression of USP8 inhibited Vif-induced A3G degradation and suppressed wild-type HIV-1 infectivity even in the presence of Vif.
PTEN deficient cells, which have low levels of USP8 and high levels of FLIP S, were relatively TRAIL resistant, and as previously noted, introduction of WT USP8 (but not blank vector or catalytically inactive USP8) increased USP8 levels and significantly increased the extent of TRAIL induced apoptosis (XREF_FIG).
Knockdown of ubpy caused trafficking defects of Wg (XREF_FIG), mimicking the mop-knockdown phenotype.
Importantly, knockdown of USP8 inhibited activation of the Akt signaling pathway by decreasing the phosphorylation level of Akt and up-regulated p53 expression, while USP8 overexpression increased activation of the Akt signaling pathway in Hucct-1 cells.
Moreover, USP8 downregulated several pro inflammatory cytokines [nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), prostaglandin E 2 (PGE 2), and interleukin-1beta (IL-1beta)] in the serum and brain, and the relevant protein factors [inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2)] in the brain.
Mechanistically, we found that USP8 increased the expression of neuregulin receptor degradation protein-1 (Nrdp1), potently downregulated the expression of TLR4 and myeloid differentiation primary response protein 88 (MyD88) protein, and inhibited the phosphorylation of IkappaB kinase (IKK) beta and kappa B-alpha (IkappaBalpha), thereby reducing nuclear translocation of p65 by inhibiting the activation of the nuclear factor-kappaB (NF-kappaB) signaling pathway in LPS induced mice.
In contrast , USP8 knockdown suppressed melanoma cell growth , survival and migration , and augmented the inhibitory effects of therapeutic drugs .
Additionally, mutant USP8, which is unable to bind to SFN, reduces the expression of RTKs and p-STAT3.
USP8 depletion causes almost complete STAM degradation and hinders further trafficking of cargo proteins 14.
Recent data suggest that USP8 may negatively regulate degradation of alpha-synuclein by removal of Lys63 ubiquitin chains.
In primary rat neurons, USP8 enhances SHANK3 and SHANK1 protein levels via deubiquitination and increases dendritic spine density.
In primary rat neurons, USP8 enhances SHANK3 and SHANK1 protein levels via deubiquitination and increases dendritic spine density.
Additionally, mutant USP8, which is unable to bind to SFN, reduces the expression of RTKs and p-STAT3.
Since Otub1was previously shown to interact with USP8, we asked whether it had any effect on USP8 modulation of GRAIL stability.
Consistently, knockdown of USP8 significantly increased apoptosis of PIK3CA mutant cells even in the presence of glutamine (XREF_SUPPLEMENTARY).
Taken together, these results indicate that USP8 mutations contribute to the pathogenesis of ACTH secreting PAs.
The inhibition of USP8 downregulated the Notch signalling pathway via NICD destabilization, resulting in the retardation of cellular growth, wound closure, and colony forming ability of breast cancer cell lines.
Here, we provide mechanistic evidence indicating that ubiquitin carboxyl-terminal hydrolase 8 (USP8) modulates BDNF and TrkB dependent neuronal differentiation.
Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.
Importantly, USP8 knock down did not impair LC3 conversion upon mTOR inhibition (XREF_SUPPLEMENTARY), further indicating that our observed effects on NCOA4 and ferritin protein levels do not reflect a general block in autophagic capacity.
A role has previously been described for USP8, in which its DUB activity directly decreases membrane LDLR downstream of IDOL by sorting it for lysosomal degradation 16.
Lysates from cells overexpressing HA-Lats2 were treated with the broad-specificity de-ubiquitylase USP8 or vehicle control and incubated with GST-YAP.
ITCH and AIP4 activity was activated by the deubiquitinases USP8 and USP9X, as demonstrated by RNA interference.
Interestingly, while USP8 inhibition and knockdown reduced levels of the phosphorylated insulin receptor, it had no effect on total levels of the protein (XREF_SUPPLEMENTARY).
Moreover, USP8 downregulated several pro inflammatory cytokines [nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), prostaglandin E 2 (PGE 2), and interleukin-1beta (IL-1beta)] in the serum and brain, and the relevant protein factors [inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2)] in the brain.
Furthermore, USP8 upregulated the anti-inflammatory mediators interleukin (IL) -4 and IL-10 in the serum and brain, and promoted a shift from pro inflammatory to anti-inflammatory microglial phenotypes.
Although it was reasonable that USP8 mutations could contribute to cell cycle dysregulation and P27 underexpression in corticotroph adenomas, our data did not confirm this hypothesis.
Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1.
Gain and loss-of-function studies showed that USP6 stabilises the membrane pool of Fzd5 and USP8 promotes the recycling of Fzd receptors to the PM, increasing their cell surface localisation and therefore enhancing Wnt signalling both in mammalian cells and Drosophila wing.
24 Deubiquitination may be necessary to efficiently target PAR2 to lysosomes because USP8 or AMSH knockdown moderately attenuated PAR2 degradation.
VEGFR2 also accumulated in EEA1 positive early endosomes when cells were treated with individual USP8 siRNAs to limit off-target effects.
Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with gamma-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation.
In zebrafish, knockout (KO) of kctd17 impairs ciliogenesis in Kupffer’s vesicle and induces situs inversus [74], whereas KO of usp8 increases ciliogenesis in the pronephric duct and causes renal cysts [50].
The results of western blot indicated that knockdown of USP8 down-regulated the expression of Cyclin D1, CDK4, CDK6, p-AKT, and Bcl2, and up-regulated the expression of Bax.
By contrast, USP8 knockdown caused the accumulation of COPII coat proteins around the cis-Golgi, promoted the intracellular trafficking of procollagen IV from the endoplasmic reticulum to the Golgi, and increased collagen IV secretion.
Moreover, silencing of USP8 also promoted apoptosis in cholangiocarcinoma cells by regulating the Bcl-2 and Bax axis and Caspase cascade; up-regulation of USP8 decreased apoptosis in Hucct-1 cells.
CLOCK deubiquitylation by USP8 inhibits CLK and CYC transcription in Drosophila.
By the same mechanism, USP8 also directs the trafficking and lysosomal degradation of CXCR4 [XREF_BIBR], MET and epidermal growth factor receptor [XREF_BIBR, XREF_BIBR], implying that its loss consequent to increased RNF41 abundance would prolong and potentially amplify invasion signaling by these receptors.
USP8 and UBPY is reported to activate the Wnt and beta-catenin pathway by targeting Frizzled G protein coupled protein XREF_BIBR.
In contrast, the expression of the wild-type or the constitutively active form USP8 S680A, but not of the catalytic mutant USP8 C748A, caused a strong reduction of the Ub-CHMP1B pool (XREF_FIG).
Overexpression of USP8 increased c-FLIP S ubiquitination, decreased c-FLIP S half-life, decreased c-FLIP S steady-state levels, and decreased TRAIL resistance.
In addition, the enlarged VEGFR2 positive endosomes did not co-distribute with late endosome marker, CD63, in cells treated with individual USP8 siRNAs.
Dex also elevated the deubiquitinating enzyme, Usp8 and Ubpy, which via Nrdp1 decreases BRUCE.
Moreover, silencing of USP8 also promoted apoptosis in cholangiocarcinoma cells by regulating the Bcl-2 and Bax axis and Caspase cascade; up-regulation of USP8 decreased apoptosis in Hucct-1 cells.
ITCH and AIP4 activity was activated by the deubiquitinases USP8 and USP9X, as demonstrated by RNA interference.
The endosome related deubiquitinating enzyme ubiquitin specific peptidase 8 (USP8) may inhibit the degradation of MVBs by regulating APP intracellular domain (AICD) protein levels [XREF_BIBR].
51) specific for USP7, HBX 90,397 inhibits USP8 (3) (Ref.
Overexpression of USP2 was also shown to reduce p53 stability [XREF_BIBR], and another de-ubiquitylase USP8 (also called UBPY) promotes epithelial growth factor receptor degradation [XREF_BIBR, XREF_BIBR, XREF_BIBR].
Given the binary protein interactions we observed among FZD, TMEM79, and USP8, we further examined how their complex assembly might occur and lead to inhibition of USP8 by TMEM79.
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Reduced MET, EGFR, and STAM2 expression was consistent with attenuation of USP8 at DUBs-IN-3 concentrations that selectively increased death in cSCC cells (XREF_FIG d).
We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs.
Further studies indicated that AUF1 protein degradation was mediated by upregulating USP8 transcription, which was modulated by its negative regulatory transcription factor Sp1.
In contrast, USP8C> S increased Smo ubiquitination, which was similar to the phenotype induced by the RNAi mediated knockdown of USP8 (XREF_FIG, lanes 2 and 4, compare to lane 1, top panel).
In Ts mice, MCS decreased expression of Becn1, Ccnf, Hspa1a, Usp8, and Rab27a (XREF_FIG), and in 2N mice of Rab11b (XREF_FIG).
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Furthermore, the presence of wild-type GRAIL along with USP8 increased the amount of ubiquitinated USP8, which was further enhanced when the DUB activity of USP8 was abolished.
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Indeed, depleting HD-PTP prevented endogenous UBPY from associating with EGFR, consistent with increased EGFR ubiquitination.
Therefore, a second process involving DUBs is the recycling of ubiquitin by preventing its degradation, which is mediated by proteasome associated DUBs USP14, UCH-L5 and UCH37, and POH1, or receptor mediated endocytosis and lysosomal degradation associated DUBs USP8 and AMSH.
Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta deficient cells.
In RPE1 cells, EGFR knockdown is sufficient to repress the USP8-trichoplein-Aurora A axis and induce ciliogenesis, however, we do not exclude the involvement of other RTKs in other cell types since USP8 is also activated by PDGFRs and FGFR1-mediated phosphorylation in vitro (Supplementary Fig. xref ).
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Then , we checked the expression of USP8 in ligament tissues from controls and patients with OPLL , and found that OPLL significantly increased the expression of USP8 ( Figure 5E ) .
Moreover, Nrdp1 overexpression augments OGD induced USP8 downregulation, while knockdown of Nrdp1 ameliorates this effect.
Stimulation of cells with the ERBB3 ligand neuregulin-1 increases Usp8 levels, which in turn stabilizes Nrdp1.
In support of this view, NGF-activated TrkA and USP8 were shown to interact on early endosomes in PC12 cells ( xref ), and activity-dependent ubiquitylation of p75 NTR was also previously reported ( xref ).
In summary, the findings in the current study highlight LRIG1 mediated Met degradation and the implication of USP8 in the regulation of LRIG1 ubiquitination by a Met targeting antibody.
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IL2R leads to the deubiquitination of ubiquitinated USP8.
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In particular, IL-2R signaling leads to Akt and mTOR activation, Otub1 translation, de-ubiquitination of ubiquitinated USP8, and subsequent degradation of GRAIL that permits T cell proliferation.
Further, IGF-1 could reverse the inhibitory effects of USP8 knockdown on the Akt signaling pathway and the proliferation of QBC939 and RBE cells.
Overexpression of Hrs or a dominant negative mutant of SKD1, proteins that play roles in the endosomal sorting of ubiquitinated receptors, caused the accumulation of endogenous UBPY on exaggerated endosomes.
HBX 90,397 , another DUB-specific inhibitor , blocks USP8 activity .
Moreover, GW7647 did not inhibit USP5 and USP8.
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Alternatively, the stoichiometry of ErbB2 induced Usp8 tyrosine phosphorylation may be lower than in the case of EGFR.
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Altogether, these data indicate that CHMP4B binds the MIT domain of UBPY and the HD-PTP Bro1 domain simultaneously, though it is formally possible that CHMP4B additionally activates UBPY MIT to bind H[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
We propose that Cbl inhibits the function of Ubpy.
In fact, the BRUCE associated deubiquitylating enzyme UBPY harbors a so called MIT-domain that mediates association with proteins of the ESCRTIII complex (Row et al., 2007).
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