Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.

A hallmark of neurodegenerative diseases is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription, and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

UBE3C restricts EV-A71 replication by ubiquitination-dependent degradation of 2C.

Ubiquitin modification of viral proteins to degrade or regulate their function is one of the strategies of the host to resist viral infection. Here, we report that ubiquitin protein ligase E3C (UBE3C), an E3 ubiquitin ligase, displayed inhibitory effects on EV-A71 replication. UBE3C knockdown resulted in increased viral protein levels and virus titers, whereas overexpression of UBE3C reduced EV-A71 replication. To explore the mechanism by which UBE3C affected EV-A71 infection, we found that the C-terminal of UBE3C bound to 2C protein and facilitated K33/K48-linked ubiquitination degradation of 2C K268. Moreover, UBE3C lost its ability to degrade 2C K268R and had a diminished inhibitory impact against the replication of recombinant EV-A71-FY-2C K268R. In addition, UBE3C also promoted ubiquitination degradation of the 2C protein of CVB3 and CVA16 and inhibited viral replication. Thus, our findings reveal a novel mechanism that UBE3C acts as an enterovirus host restriction factor, including EV-A71, by targeting the 2C protein. IMPORTANCE: The highly conserved 2C protein of EV-A71 is a multifunctional protein and plays a key role in the replication cycle. In this study, we demonstrated for the first time that UBE3C promoted the degradation of 2C K268 via K33/K48-linked ubiquitination, thereby inhibiting viral proliferation. Our findings advance the knowledge related to the roles of 2C in EV-A71 virulence and the ubiquitination pathway in the host restriction of EV-A71 infection.

Nervous necrosis virus capsid protein and Protein A dynamically modulate the fish cGAS-mediated IFN signal pathway to facilitate viral evasion.

Nervous necrosis virus (NNV), an aquatic RNA virus belonging to Betanodavirus, infects a variety of marine and freshwater fishes, leading to massive mortality of cultured larvae and juveniles and substantial economic losses. The enzyme cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) is widely recognized as a central component in the innate immune response to cytosolic DNA derived from different pathogens. However, little is known about the response of cGAS to aquatic RNA viruses. This study found that Epinephelus coioides cGAS (EccGAS) overexpression inhibited NNV replication, whereas EccGAS silencing promoted NNV replication. The anti-NNV activity of EccGAS was involved in interferon (IFN) signaling activation including tumor necrosis factor receptor-associated factor family member-associated NF-kappa-B activator-binding kinase 1 (TBK1) phosphorylation, interferon regulatory factor 3 (IRF3) nuclear translocation, and the subsequent induction of IFNc and ISGs. Interestingly, NNV employed its capsid protein (CP) or Protein A (ProA) to negatively or positively modulate EccGAS-mediated IFN signaling by simultaneously targeting EccGAS. CP interacted with EccGAS via the arm-P, S-P, and SD structural domains and promoted its polyubiquitination with K48 and K63 linkages in an EcUBE3C (the ubiquitin ligase)-dependent manner, ultimately leading to EccGAS degradation. Conversely, ProA bound to EccGAS and inhibited its ubiquitination and degradation. In regulating EccGAS protein content, CP's inhibitory action was more pronounced than ProA's protective effect, allowing successful NNV replication. These novel findings suggest that NNV CP and ProA dynamically modulate the EccGAS-mediated IFN signaling pathway to facilitate the immune escape of NNV. Our findings shed light on a novel mechanism of virus-host interaction and provide a theoretical basis for the prevention and control of NNV.IMPORTANCEAs a well-known DNA sensor, cGAS is a pivotal component in innate anti-viral immunity to anti-DNA viruses. Although there is growing evidence regarding the function of cGAS in the resistance to RNA viruses, the mechanisms by which cGAS participates in RNA virus-induced immune responses in fish and how aquatic viruses evade cGAS-mediated immune surveillance remain elusive. Here, we investigated the detailed mechanism by which EccGAS positively regulates the anti-NNV response. Furthermore, NNV CP and ProA interacted with EccGAS, regulating its protein levels through ubiquitin-proteasome pathways, to dynamically modulate the EccGAS-mediated IFN signaling pathway and facilitate viral evasion. Notably, NNV CP was identified to promote the ubiquitination of EccGAS via ubiquitin ligase EcUBE3C. These findings unveil a novel strategy for aquatic RNA viruses to evade cGAS-mediated innate immunity, enhancing our understanding of virus-host interactions.

UBE3C tunes autophagy via ATG4B ubiquitination.

ATG4B is a core protein and essential for cleaving precursor MAP1LC3/LC3 or deconjugating lipidated LC3-II to drive the formation of autophagosomes. The protein stability and activity of ATG4B regulated by post-translational modification (ubiquitination) will directly affect macroautophagy/autophagy. However, the mechanism involved in ATG4B ubiquitination is largely unclear. In this study, a new E3 ligase of ATG4B, UBE3C, was identified by mass spectra. UBE3C mainly assembles K33-branched ubiquitin chains on ATG4B at Lys119 without causing ATG4B degradation. In addition, the increased ubiquitination of ATG4B caused by UBE3C overexpression inhibits autophagy flux in both normal and starvation conditions, which might be due to the reduced activity of ATG4B and ATG4B-LC3 interaction. This reduction could be reversed once the lysine 119 of ATG4B was mutated to arginine. More important, under starvation conditions the interaction between ATG4B and UBE3C apparently decreased followed by the removal of the K33-branched ubiquitin chain of ATG4B. Thus, starvation-induced autophagy could be partially suppressed by an increased ubiquitination level of ATG4B. In conclusion, our research reveals a novel modification mode of ATG4B in which UBE3C can fine tune ATG4B activity by specific ubiquitination regulating autophagy without causing ATG4B degradation.Abbreviation: ATG: autophagy-related; Baf: bafilomycin A1; CBB: Coomassie Brilliant Blue; CM: complete medium; CQ: chloroquine; GFP: green fluorescent protein; HA-Ub: HA-tagged ubiquitin; IF: immunofluorescence; IP: immunoprecipitation; K: lysine; KO: knockout; K0: all K-to-R mutant; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MS: mass spectrometry; NC: negative control; R: arginine; WCL: whole cell lysate; WT: wild-type.

UBE3C Facilitates the ER-Associated and Peripheral Degradation of Misfolded CFTR.

The ubiquitin E3 ligase UBE3C promotes the proteasomal degradation of cytosolic proteins and endoplasmic reticulum (ER) membrane proteins. UBE3C is proposed to function downstream of the RNF185/MBRL ER-associated degradation (ERAD) branch, contributing to the ERAD of select membrane proteins. Here, we report that UBE3C facilitates the ERAD of misfolded CFTR, even in the absence of both RNF185 and its functional ortholog RNF5 (RNF5/185). Unlike RNF5/185, UBE3C had a limited impact on the ubiquitination of misfolded CFTR. UBE3C knockdown (KD) resulted in an additional increase in the functional ∆F508-CFTR channels on the plasma membrane when combined with the RNF5/185 ablation, particularly in the presence of clinically used CFTR modulators. Interestingly, although UBE3C KD failed to attenuate the ERAD of insig-1, it reduced the ERAD of misfolded ∆Y490-ABCB1 and increased cell surface expression. UBE3C KD also stabilized the mature form of ∆F508-CFTR and increased the cell surface level of T70-CFTR, a class VI CFTR mutant. These results suggest that UBE3C plays a vital role in the ERAD of misfolded CFTR and ABCB1, even within the RNF5/185-independent ERAD pathway, and it may also be involved in maintaining the peripheral quality control of CFTR.

LINC00963-FOSB-mediated transcription activation of UBE3C enhances radioresistance of breast cancer cells by inducing ubiquitination-dependent protein degradation of TP73.

BACKGROUND: The ubiquitin protein ligase E3C (UBE3C) has been reported to play an oncogenic role in breast cancer (BRCA). This work further investigates the effect of UBE3C on the radioresistance of BRCA cells. METHODS: Molecules linking to radioresistance in BRCA were identified by analyzing two GEO datasets, GSE31863 and GSE101920. UBE3C overexpression or knockdown was induced in parental or radioresistant BRCA cells, followed by irradiation treatment. The malignant properties of cells in vitro, and the growth and metastatic activity of cells in nude mice, were analyzed. Downstream target proteins, as well as upstream transcriptional regulators of UBE3C, were predicted by bioinformatics tools. Molecular interactions were confirmed by immunoprecipitation and immunofluorescence assays. Furthermore, artificial alterations of TP73 and FOSB were induced in the BRCA cells for functional rescue assays. RESULTS: According to bioinformatics analyses, UBE3C expression was linked to radioresistance in BRCA. UBE3C knockdown in radioresistant BRCA cells reduced while its overexpression in parental BRCA cells increased the radioresistance of cells in vitro and in vivo. UBE3C, which induced ubiquitination-dependent protein degradation of TP73, was transcriptionally activated by FOSB. The radioresistance of cancer cells was blocked by TP73 overexpression or FOSB knockdown. Additionally, LINC00963 was found to be responsible for the recruitment of FOSB to the UBE3C promoter for transcription activation. CONCLUSION: This work demonstrates that LINC00963 induces nuclear translocation of FOSB and the consequent transcription activation of UBE3C, which enhances radioresistance of BRCA cells by inducing ubiquitination-dependent protein degradation of TP73.

Biallelic variants in HECT E3 paralogs, HECTD4 and UBE3C, encoding ubiquitin ligases cause neurodevelopmental disorders that overlap with Angelman syndrome.

PURPOSE: Pathogenic variants in genes encoding ubiquitin E3 ligases are known to cause neurodevelopmental syndromes. Additional neurodevelopmental disorders associated with the other genes encoding E3 ligases are yet to be identified. METHODS: Chromosomal analysis and exome sequencing were used to identify the genetic causes in 10 patients from 7 unrelated families with syndromic neurodevelopmental, seizure, and movement disorders and neurobehavioral phenotypes. RESULTS: In total, 4 patients were found to have 3 different homozygous loss-of-function (LoF) variants, and 3 patients had 4 compound heterozygous missense variants in the candidate E3 ligase gene, HECTD4, that were rare, absent from controls as homozygous, and predicted to be deleterious in silico. In 3 patients from 2 families with Angelman-like syndrome, paralog-directed candidate gene approach detected 2 LoF variants in the other candidate E3 ligase gene, UBE3C, a paralog of the Angelman syndrome E3 ligase gene, UBE3A. The RNA studies in 4 patients with LoF variants in HECTD4 and UBE3C provided evidence for the LoF effect. CONCLUSION: HECTD4 and UBE3C are novel biallelic rare disease genes, expand the association of the other HECT E3 ligase group with neurodevelopmental syndromes, and could explain some of the missing heritability in patients with a suggestive clinical diagnosis of Angelman syndrome.

P38α MAPK is a gatekeeper of uterine progesterone responsiveness at peri-implantation via Ube3c-mediated PGR degradation.

Estrogen and progesterone specify the establishment of uterine receptivity mainly through their respective nuclear receptors, ER and PR. PR is transcriptionally induced by estrogen-ER signaling in the endometrium, but how the protein homeostasis of PR in the endometrium is regulated remains elusive. Here, we demonstrated that the uterine-selective depletion of P38α derails normal uterine receptivity ascribed to the dramatic down-regulation of PR protein and disordered progesterone responsiveness in the uterine stromal compartment, leading to defective implantation and female infertility. Specifically, Ube3c, an HECT family E3 ubiquitin ligase, targets PR for polyubiquitination and thus proteasome degradation in the absence of P38α. Moreover, we discovered that P38α restrains the polyubiquitination activity of Ube3c toward PR by phosphorylating the Ube3c at serine741 . In summary, we provided genetic evidence for the regulation of PR protein stability in the endometrium by P38α and identified Ube3c, whose activity was modulated by P38α-mediated phosphorylation, as an E3 ubiquitin ligase for PR in the uterus.

Circular RNA circPOLR2A promotes clear cell renal cell carcinoma progression by facilitating the UBE3C-induced ubiquitination of PEBP1 and, thereby, activating the ERK signaling pathway.

BACKGROUND: Increasing evidence has demonstrated that circular RNAs (circRNAs) are implicated in cancer progression. However, the aberrant expression and biological functions of circRNAs in clear cell renal cell carcinoma (cRCC) remain largely elusive. METHOD: Differentially expressed circRNAs in cRCC were filtered via bioinformatics analysis. Aberrant circPOLR2A expression was validated in cRCC tissues and cell lines via qRT-PCR. Sanger sequencing was used to identify the backsplicing site of circPOLR2A. In vitro and in vivo functional experiments were performed to evaluate the role of circPOLR2A in cRCC malignancy. RNA pull-down, mass spectrometry, RIP, FISH and immunofluorescence assays were used to identify and validate the circPOLR2A-interacting proteins. Ubiquitination modification and interaction between proteins were detected via Co-IP and western blotting. The m6A modification in circPOLR2A was validated by the meRIP assay. RESULTS: Bioinformatics analysis revealed that circPOLR2A was highly expressed in cRCC tissues and metastatic cRCC tissues. CircPOLR2A expression was associated with tumor size and TNM stage in cRCC patients. In vitro and in vivo functional assays revealed that circPOLR2A accelerated cRCC cell proliferation, migration, invasion and angiogenesis, while inhibiting apoptosis. Further mechanistic research suggested that circPOLR2A could interact with UBE3C and PEBP1 proteins, and that UBE3C could act as a specific ubiquitin E3 ligase for the PEBP1 protein. The UBE3C/circPOLR2A/PEBP1 protein-RNA ternary complex enhanced the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein which could inactivate the ERK signaling pathway. Rescue experiments revealed that the PEBP1 protein was the functional downstream target of circPOLR2A. Furthermore, m6A modification in circPOLR2A was confirmed, and the m6A reader YTHDF2 could regulate circPOLR2A expression. CONCLUSION: Our study demonstrated that circPOLR2A modulated the UBE3C-mediated ubiquitination and degradation of the PEBP1 protein, and further activated the ERK pathway during cRCC progression and metastasis. The m6A reader, YTHDF2, regulated circPOLR2A expression in cRCC. Hence, circPOLR2A could be a potential target for the diagnosis and treatment of cRCC.

DOT1L O-GlcNAcylation promotes its protein stability and MLL-fusion leukemia cell proliferation.

Histone lysine methylation functions at the interface of the extracellular environment and intracellular gene expression. DOT1L is a versatile histone H3K79 methyltransferase with a prominent role in MLL-fusion leukemia, yet little is known about how DOT1L responds to extracellular stimuli. Here, we report that DOT1L protein stability is regulated by the extracellular glucose level through the hexosamine biosynthetic pathway (HBP). Mechanistically, DOT1L is O-GlcNAcylated at evolutionarily conserved S1511 in its C terminus. We identify UBE3C as a DOT1L E3 ubiquitin ligase promoting DOT1L degradation whose interaction with DOT1L is susceptible to O-GlcNAcylation. Consequently, HBP enhances H3K79 methylation and expression of critical DOT1L target genes such as HOXA9/MEIS1, promoting cell proliferation in MLL-fusion leukemia. Inhibiting HBP or O-GlcNAc transferase (OGT) increases cellular sensitivity to DOT1L inhibitor. Overall, our work uncovers O-GlcNAcylation and UBE3C as critical determinants of DOT1L protein abundance, revealing a mechanism by which glucose metabolism affects malignancy progression through histone methylation.

Oncogenic UBE3C promotes breast cancer progression by activating Wnt/ß-catenin signaling.

BACKGROUND: Breast cancer (BrCa) is the most common female malignancy worldwide and has the highest morbidity among all cancers in females. Unfortunately, the mechanisms of BrCa growth and metastasis, which lead to a poor prognosis in BrCa patients, have not been well characterized. METHODS: Immunohistochemistry (IHC) was performed on a BrCa tissue microarray (TMA) containing 80 samples to evaluate ubiquitin protein ligase E3C (UBE3C) expression. In addition, a series of cellular experiments were conducted to reveal the role of UBE3C in BrCa. RESULTS: In this research, we identified UBE3C as an oncogenic factor in BrCa growth and metastasis for the first time. UBE3C expression was upregulated in BrCa tissues compared with adjacent breast tissues. BrCa patients with high nuclear UBE3C expression in tumors showed remarkably worse overall survival (OS) than those with low nuclear expression. Knockdown of UBE3C expression in MCF-7 and MDA-MB-453 BrCa cells inhibited cell proliferation, migration and invasion in vitro, while overexpression of UBE3C in these cells exerted the opposite effects. Moreover, UBE3C promoted ß-catenin nuclear accumulation, leading to the activation of the Wnt/ß-catenin signaling pathway in BrCa cells. CONCLUSION: Collectively, these results imply that UBE3C plays crucial roles in BrCa development and progression and that UBE3C may be a novel target for the prevention and treatment of BrCa.

Association of UBE3C Variants with Reduced Kidney Function in Patients with Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is the leading cause of morbidity and mortality in patients with diabetes mellitus (DM) and the most common variant of end-stage renal disease (ESRD) globally. The economic burden of ESRD treatment with dialysis is substantial. The incidence and prevalence of ESRD in Taiwan remain the highest worldwide. Therefore, identifying genetic factors affecting kidney function would have valuable clinical implications. We performed microarray experiments and identified that ubiquitin protein ligase E3C (UBE3C) is differentially expressed in two DKD patient groups with extreme (low and high) urine protein-to-creatinine ratios. A follow-up genotyping study was performed in a larger group to investigate any specific variants of UBE3C associated with DKD. A total of 263 patients were included in the study, comprising 172 patients with DKD and 91 control subjects (patients with DM without chronic kidney disease (CKD)). Two UBE3C variants (rs3802129(AA) and rs7807(CC)) were determined to be associated with reduced kidney function. The haplotype analysis revealed that rs3802129/rs3815217 (block 1) with A/G haplotype and rs8101/rs7807 (block 2) with T/C haplotype were associated with higher risks of CKD phenotypes. These findings suggest a clinical role of UBE3C variants in DKD risk.

Molecular Evolution, Neurodevelopmental Roles and Clinical Significance of HECT-Type UBE3 E3 Ubiquitin Ligases.

Protein ubiquitination belongs to the best characterized pathways of protein degradation in the cell; however, our current knowledge on its physiological consequences is just the tip of an iceberg. The divergence of enzymatic executors of ubiquitination led to some 600-700 E3 ubiquitin ligases embedded in the human genome. Notably, mutations in around 13% of these genes are causative of severe neurological diseases. Despite this, molecular and cellular context of ubiquitination remains poorly characterized, especially in the developing brain. In this review article, we summarize recent findings on brain-expressed HECT-type E3 UBE3 ligases and their murine orthologues, comprising Angelman syndrome UBE3A, Kaufman oculocerebrofacial syndrome UBE3B and autism spectrum disorder-associated UBE3C. We summarize evolutionary emergence of three UBE3 genes, the biochemistry of UBE3 enzymes, their biology and clinical relevance in brain disorders. Particularly, we highlight that uninterrupted action of UBE3 ligases is a sine qua non for cortical circuit assembly and higher cognitive functions of the neocortex.

Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex.

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.

Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes.

The intracellular accumulation of aggregated misfolded proteins is a cytopathological hallmark of neurodegenerative diseases. However, the functional relationship between protein misfolding or aggregation and the cellular proteostasis network that monitors and maintains proteome health is poorly understood. Previous studies have associated translational suppression and transcriptional remodeling with the appearance of protein aggregates, but whether these responses are induced by aggregates or their misfolded monomeric or oligomeric precursors remains unclear. Because aggregation in cells is rapid, nonlinear, and asynchronous, it has not been possible to deconvolve these kinetically linked processes to determine the earliest cellular responses to misfolded proteins. Upon removal of the synthetic, biologically inert ligand shield-1 (S1), AgDD, an engineered variant FK506-binding protein (FKBP1A), rapidly (t½ ∼5 min) unfolds and self-associates, forming detergent-insoluble, microscopic cytoplasmic aggregates. Using global diglycine-capture (K-GG) proteomics, we found here that this solubility transition is associated with immediate increases in ubiquitylation of AgDD itself, along with that of endogenous proteins that are components of the ribosome and the 26S proteasome. We also found that the earliest cellular responses to acute S1 removal include recruitment of ubiquitin protein ligase E3C (UBE3C) to the 26S proteasome and ubiquitylation of two key proteasomal ubiquitin receptors, 26S proteasome regulatory subunit RPN10 (RPN10) and Rpn13 homolog (RPN13 or ADRM1). We conclude that these proteasomal responses are due to AgDD protein misfolding and not to the presence of detergent-insoluble aggregates.

MiR-30a-5p/UBE3C axis regulates breast cancer cell proliferation and migration.

Aberrant expression of ubiquitin Protein ligase E3C (UBE3C) has been documented in breast cancer (BC). MicroRNAs (miRNAs) were shown to play an important role in the regulation of tumor properties in BC. However, whether miRNAs contributes to UBE3C expression in BC cells remains poorly understood. In this study, we report that UBE3C was a direct target of miR-30a-5p. Expression of miR-30a-5p in BC cells reduced UBE3C expression. MCF-7 and MDA-MB-453 cells were transfected miR-30a-5p-overexpression, and found that cell proliferation and migration were inhibited. In contrast, when miR-30a-5p inhibitor were transfected into MCF-7 and MDA-MB-453 cells, cell proliferation and migration were promoted. We study demonstrated that upregulation of miR-30a-5p was significantly suppressed levels of cyclin B1, cyclin D1 and c-myc. Moreover, Correlation analysis indicated that expression of miR-30a-5p was highly negatively correlated with UBE3C, which was upregulated in BC specimens. These data highlight the important role of miR-30a-5p/UBE3C axis in BC development and progression. Therefore, miR-30a-5p activation or UBE3C inhibition may be provide a novel strategy for the treatment of BC.

Effects of UBE3C on proliferation and invasion in ovarian cancer SKOV3 cells / 上海交通大学学报（医学版）

Objective • To explore the effects of ubiquitin protein ligase E3C (UBE3C) on proliferation and invasion in epithelial ovarian cancer SKOV3 cells. Methods • Western blotting was used to detect the expression difference of UBE3C in epithelial ovarian cancer cell lines and normal ovarian cell lines. SKOV3 cells were transfected with si-UBE3C to knockdown UBE3C protein level, while ex-UBE3C plasmid was used to upregulate the expression of UBE3C. Cell proliferation was measured by CCK-8 assay and colony formation assay. Wound healing assay and Transwell assay were performed to investigate the effect of UBE3C on migration and invasion. Protein levels of β-catenin and c-Myc were also detected in different groups, which were closely related to proliferation and invasion. Results • UBE3C was highly expressed in epithelial ovarian cancer cell lines. UBE3C was successfully silenced with si-UBE3C transfection in SKOV3 cells. Inhibition of UBE3C significantly weakened the abilities of cell proliferation, migration and invasion. A reduction of β-catenin and c-Myc protein levels was also accompanied by UBE3C knockdown. Overexpression of UBE3C with ex- UBE3C plasmid promoted the abilities of proliferation, migration and invasion. Enhanced expression levels of β-catenin and c-Myc were also verified. Conclusion • UBE3C promotes proliferation and invasion of epithelial ovarian cancer SKOV3 cells. This might be to do with upregulation of β-catenin and c-Myc protein levels.

Effects of UBE3C on proliferation and invasion in ovarian cancer SKOV3 cells / 上海交通大学学报（医学版）

Objective·To explore the effects of ubiquitin protein ligase E3C (UBE3C) on proliferation and invasion in epithelial ovarian cancer SKOV3 cells. Methods?·?Western blotting was used to detect the expression difference of UBE3C in epithelial ovarian cancer cell lines and normal ovarian cell lines. SKOV3 cells were transfected with si-UBE3C to knockdown UBE3C protein level, while ex-UBE3C plasmid was used to upregulate the expression of UBE3C. Cell proliferation was measured by CCK-8 assay and colony formation assay. Wound healing assay and Transwell assay were performed to investigate the effect of UBE3C on migration and invasion. Protein levels of β-catenin and c-Myc were also detected in different groups, which were closely related to proliferation and invasion. Results?·?UBE3C was highly expressed in epithelial ovarian cancer cell lines. UBE3C was successfully silenced with si-UBE3C transfection in SKOV3 cells. Inhibition of UBE3C significantly weakened the abilities of cell proliferation, migration and invasion. A reduction of β-catenin and c-Myc protein levels was also accompanied by UBE3C knockdown. Overexpression of UBE3C with ex-UBE3C plasmid promoted the abilities of proliferation, migration and invasion. Enhanced expression levels of β-catenin and c-Myc were also verified. Conclusion?·?UBE3C promotes proliferation and invasion of epithelial ovarian cancer SKOV3 cells. This might be to do with upregulation of β-catenin and c-Myc protein levels.

MiR-542-3p inhibits metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma by targeting UBE3C.

Accumulating evidence demonstrates that aberrant miRNAs contribute to hepatocellular carcinoma (HCC) development and progression. However, the roles of various miRNAs in HCC remain to be determined. In present research, we confirmed that a reduced miR-542-3p expression was present in HCC tissues and cell lines. Our clinical analysis revealed that the down-regulated miR-542-3p expression was significantly correlated with poor prognostic features including advanced TNM stage and venous infiltration. Moreover, we confirmed that miR-542-3p was a novel independent prognostic marker for predicting 5-year survival of HCC patients. The ectopic overexpression of miR-542-3p inhibited cell migration, invasion and EMT progress, while down-regulated miR-542-3p reversed the effect. In addition, miR-542-3p could regulate UBE3C by directly binding to its 3'-UTR. In clinical samples of HCC, miR-542-3p inversely correlated with UBE3C, which was upregulated in HCC. Alternation of UBE3C expression at least partially abolished the migration, invasion and EMT progress effects of miR-542-3p on HCC cells. In conclusion, our results indicated that miR-542-3p functioned as a tumor suppressor gene in regulating the EMT and metastasis of HCC via targeting UBE3C, and may represent a novel potential therapeutic target and prognostic marker for HCC.

Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c.

In mammalian cells, the 26S proteasomes vary in composition. In addition to the standard 28 subunits in the 20S core particle and 19 subunits in each 19S regulatory particle, a small fraction (about 10-20% in our preparations) also contains the deubiquitinating enzyme Usp14/Ubp6, which regulates proteasome activity, and the ubiquitin ligase, Ube3c/Hul5, which enhances proteasomal processivity. When degradation of ubiquitinated proteins in cells was inhibited, levels of Usp14 and Ube3c on proteasomes increased within minutes. Conversely, when protein ubiquitination was prevented, or when purified proteasomes hydrolyzed the associated ubiquitin conjugates, Usp14 and Ube3c dissociated rapidly (unlike other 26S subunits), but the inhibitor ubiquitin aldehyde slowed their dissociation. Recombinant Usp14 associated with purified proteasomes preferentially if they contained ubiquitin conjugates. In cells or extracts, adding Usp14 inhibitors (IU-1 or ubiquitin aldehyde) enhanced Usp14 and Ube3c binding further. Thus, in the substrate- or the inhibitor-bound conformations, Usp14 showed higher affinity for proteasomes and surprisingly enhanced Ube3c binding. Moreover, adding ubiquitinated proteins to cell extracts stimulated proteasome binding of both enzymes. Thus, Usp14 and Ube3c cycle together on and off proteasomes, and the presence of ubiquitinated substrates promotes their association. This mechanism enables proteasome activity to adapt to the supply of substrates.