Ubiquitin Protein Ligase E3C Research Articles

M6A writer gene fl(2)d regulates the metamorphosis of silkworm Bombyx mori

The N6-methyladenosine (m6A) is one of the most abundant posttranscriptional modifications on RNAs, which is mediated by its effector proteins—writers, readers, and erasers. Female-Lethal(2)D (Fl(2)d) is one of the key writers to stabilize the interaction between another two writers Methyltransferase-like 3 (METTL3) and Methyltransferase-like 14 (METTL14). Here with dsRNA transfection, fl(2)d was knocked down in the BmN4 cell lines of silkworm, the expression of 61 genes were significantly increased, and 86 genes were significantly decreased after fl(2)d repression, which encoded proteins related to catalytic, hydrolase activity or in metabolism of nucleic acid. To elucidate the functional role of fl(2)d during silkworm development, dsRNA of which was injected into the hemolymph of the 2-d-old fourth instar larvae, the mounting process of silkworm was delayed, the expression level of genes in metabolism, RNA processing and ecdysone signaling pathway decreased significantly after fl(2)d knockdown, such as genes encoded Ubiquitin protein ligase E3C, the nuclear pore complex protein Nup98-Nup96, and the m6A reader YTHDF3, as well as Broad Complex isoform 2 (BRC-Z2), Fushi Tarazu Factor-1 (FTZ-F1), lactate dehydrogenase (LDH) and the ecdysone-induced protein 74EF isoform A (E74A). Total m6A level of RNAs also decreased after fl(2)d repression. These indicated that decreased level of fl(2)d and YTHDF3 decay the m6A level and translation of their target genes, which might lead to the retardation of metamorphosis, and resulted in the delayed mounting of silkworm.

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

BackgroundThe 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.MethodsMolecules 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.ResultsAccording 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.ConclusionThis 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.Graphical

Tegument protein UL21 of alpha-herpesvirus inhibits the innate immunity by triggering CGAS degradation through TOLLIP-mediated selective autophagy

ABSTRACT Alpha-herpesvirus causes lifelong infections and serious diseases in a wide range of hosts and has developed multiple strategies to counteract the host defense. Here, we demonstrate that the tegument protein UL21 (unique long region 21) in pseudorabies virus (PRV) dampens type I interferon signaling by triggering the degradation of CGAS (cyclic GMP-AMP synthase) through the macroautophagy/autophagy-lysosome pathway. Mechanistically, the UL21 protein scaffolds the E3 ligase UBE3C (ubiquitin protein ligase E3C) to catalyze the K27-linked ubiquitination of CGAS at Lys384, which is recognized by the cargo receptor TOLLIP (toll interacting protein) and degraded in the lysosome. Additionally, we show that the N terminus of UL21 in PRV is dominant in destabilizing CGAS-mediated innate immunity. Moreover, viral tegument protein UL21 in herpes simplex virus type 1 (HSV-1) also displays the conserved inhibitory mechanisms. Furthermore, by using PRV, we demonstrate the roles of UL21 in degrading CGAS to promote viral infection in vivo. Altogether, these findings describe a distinct pathway where alpha-herpesvirus exploits TOLLIP-mediated selective autophagy to evade host antiviral immunity, highlighting a new interface of interplay between the host and DNA virus. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AHV-1: anatid herpesvirus 1; ATG7: autophagy related 7; ATG13: autophagy related 13; ATG101: autophagy related 101; BHV-1: bovine alphaherpesvirus 1; BNIP3L/Nix: BCL2 interacting protein 3 like; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCDC50: coiled-coil domain containing 50; CCT2: chaperonin containing TCP1 subunit 2; CGAS: cyclic GMP-AMP synthase; CHV-2: cercopithecine herpesvirus 2; co-IP: co-immunoprecipitation; CQ: chloroquine; CRISPR: clustered regulatory interspaced short palindromic repeat; Cas9: CRISPR-associated system 9; CTD: C-terminal domain; Ctrl: control; DAPI: 4’,6-diamidino-2-phenylindole; DBD: N-terminal DNA binding domain; DMSO: dimethyl sulfoxide; DYNLRB1: dynein light chain roadblock-type 1; EHV-1: equine herpesvirus 1; gB: glycoprotein B; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HSV-1: herpes simplex virus 1; HSV-2: herpes simplex virus 2; IB: immunoblotting; IRF3: interferon regulatory factor 3; lenti: lentivirus; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARCHF9: membrane associated ring-CH-type finger 9; MG132: cbz-leu-leu-leucinal; NBR1: NBR1 autophagy cargo receptor; NC: negative control; NEDD4L: NEDD4 like E3 ubiquitin protein ligase; NH4Cl: ammonium chloride; OPTN: optineurin; p-: phosphorylated; PFU: plaque-forming unit; Poly(dA:dT): Poly(deoxyadenylic-deoxythymidylic) acid; PPP1: protein phosphatase 1; PRV: pseudorabies virus; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RNF126: ring finger protein 126; RT-PCR: real-time polymerase chain reaction; sgRNA: single guide RNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; TOLLIP: toll interacting protein; TRIM33: tripartite motif containing 33; UL16: unique long region 16; UL21: unique long region 21; UL54: unique long region 54; Ub: ubiquitin; UBE3C: ubiquitin protein ligase E3C; ULK1: unc-51 like autophagy activating kinase 1; Vec: vector; VSV: vesicular stomatitis virus; VZV: varicella-zoster virus; WCL: whole-cell lysate; WT: wild-type; Z-VAD: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone.

Oncogenic UBE3C promotes breast cancer progression by activating Wnt/\u03b2-catenin signaling

BackgroundBreast 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.MethodsImmunohistochemistry (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.ResultsIn 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 β-catenin nuclear accumulation, leading to the activation of the Wnt/β-catenin signaling pathway in BrCa cells.ConclusionCollectively, 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.

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.

Ubiquitin-protein ligase E3C maintains non-small-cell lung cancer stemness by targeting AHNAK-p53 complex

Cancer stem-like cells (CSCs) are regarded as sources of tumorigenesis, metastasis, and drug resistance, which limits current cancer therapies. Elucidating the molecular modes governing CSC properties is necessary to optimize therapeutic approaches. In this study, we discovered that ubiquitin-protein ligase E3C (UBE3C)-mediated ubiquitination is a key posttranslational mechanism involved in maintaining CSC properties of non-small-cell lung cancer (NSCLC). UBE3C was overexpressed in stem-like NSCLC cells and acted as a stemness enhancer. Knockdown of UBE3C reduced NSCLC stemness and tumorigenesis both in vivo and in vitro. We further identified AHNAK as a novel UBE3C substrate, finding that UBE3C maintained stemness by ubiquitinating and promoting AHNAK degradation. AHNAK functioned as a cofactor assisting p53 binding to stemness-related gene promoters to inhibit transcription. Subsequent downregulation of AHNAK by UBE3C overexpression removed p53-mediated inhibition of gene expression, resulting in enhanced stemness. Clinical significance was investigated in 208 NSCLC patients and confirmed that attenuated UBE3C activity and elevated AHNAK protein levels correlated with extended survival time. Collectively, findings reveal the first global characterization of UBE3C-mediated ubiquitination as a key regulator of CSCs, with results suggesting involvement of the AHNAK-p53 complex.

PO-511 High miR-9 levels represent a novel prognostic biomarker to predict development of malignant meningioma

IntroductionMeningioma is the most common primary tumour affecting the central nervous system; it is classified as benign (WHO I,~80%), atypical (WHO II,~15%–20%) and anaplastic/malignant (WHO III,~1%–3%). The 3 year recurrence rate in WHO I meningioma is estimated in about 50% and it is much greater in WHO II and III tumours. MicroRNAs (miRNAs) represent a large class of small RNAs driving regulation of gene expression at post-transcriptional level and playing a role in cell proliferation, differentiation, apoptosis and carcinogenesis. Several studies showed that miRNAs are involved in tumour progression and therefore proposed as diagnostic tools. Here, we evaluated miRNAs signature in meningioma to identify novel biomarkers of tumour progression.Material and methodsMeningioma (MN) specimens were collected from consented patients, according to the ethics. The 96-miRNA profiling was performed using the QuantimirTM Cancer MicroRNA qPCR Array (System Biosciences, UK) following the instructions of the supplier. Validation studies were achieved using TaqMan MicroRNA reagents (Applied Biosystems). Bioinformatic analysis was done using the NormFinder software and in silico studies were performed using the following datasets for putative miRNA targets: TargetScanHuman7.1, DIANATOOLS microT-CDS, mirDIP and the UTRdb tool. Probability (p) values were calculated using the Student’s t-Test, led by the GraphPad Prism 5.01 (p<0.05±SEM).Results and discussionsWe established a new miRNA dataset by identifying six miRNA signatures (p<0.01) differentially regulated in benign versus malignant meningioma cells (miR-9, −10b, −125b, −143,–145 and −199). Validation studies by qPCR confirmed that the miR-9 was upregulated in malignant KT21-MG1 cells (10.71 folds; Log2 scale, p=0.0006) and WHO III tissues (3.75 folds versus WHO I=0, Log2 scale, p=0.044). Finally, highly stringent in silico studies suggested that the miR-9 targets and downregulates the ubiquitin-protein ligase E3C (UBE3C), as confirmed by proteomic analysis in malignant KT21-MG1 and IOMM-Lee cells.ConclusionIn this study, we identified the miR-9 as significantly upregulated in WHO III tumour-derived meningioma cells and tissues when compared to lower grades. Since miR-9 targets the ubiquitin-protein ligase E3C, involved in the ubiquitin-proteasome pathway and therefore regulating protein homeostasis, this protein, together with miR-9, could represent potential novel diagnostic and/or prognostic biomarkers in meningioma.

Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification.

Ossification of the posterior longitudinal ligament (OPLL), a spinal ligament, reduces the range of motion in limbs. No treatment is currently available for OPLL, which is why therapies are urgently needed. OPLL occurs in obesity, is more common in men, and has an onset after 40 years of age. The mechanisms underlying OPLL remain unclear. In this study, we performed a serum proteomic analysis in both OPLL patients and healthy subjects to identify factors potentially involved in the development of OPLL, and found reduced levels of a protein that might underlie the pathology of OPLL. We isolated the protein, determined its amino acid sequence, and identified it as chemokine (C-X-C motif) ligand 7 (CXCL7). Based on these proteomics findings, we generated a CXCL7 knockout mouse model to study the molecular mechanisms underlying OPLL. CXCL7-null mice presented with a phenotype of OPLL, showing motor impairment, heterotopic ossification in the posterior ligament tissue, and osteoporosis in vertebrate tissue. To identify the mechanisms of CXCL7 deficiency in OPLL, we searched for single nucleotide polymorphisms and altered DNA exons, but no abnormalities were found. Although miR-340 levels were found to be high in an miRNA array, they were insufficient to reduce CXCL7 levels. Ubiquitin C-terminal hydrolase1 (UCHL1) was found to be overexpressed in CXCL7-null mice and in the sera of patients with OPLL, and was correlated with OPLL severity. Post-translational modifications of proteins with ubiquitin and ubiquitin-like modifiers, orchestrated by a cascade of specialized ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (E3) enzymes, are thought to control a wide range of cellular processes, and alterations in the ubiquitin–proteasome system have been associated with several degenerative disorders. In addition, the OPLL tissue of CXCL7-null mouse and its primary cells expressed the antibody to ubiquitin (linkage-specific K48). Our data clearly show decreased CXCL7 levels in patients with OPLL, and that OPLL developed in mice lacking CXCL7. Tumor necrosis factor receptor-associated factor (TRAF)6 expression was decreased in CXCL7-null mouse primary cells. Furthermore, K48 polyubiquitination was found in posterior longitudinal ligament ossified tissue and primary cells from CXCL7-null mice. We performed a phosphoproteomics analysis in CXCL7-deficient mice and identified increased phosphorylation of mitogen-activated protein kinase kinase (ME3K)15, ubiquitin protein ligase E3C (UBE3C) and protein kinase C (PKC) alpha, suggesting that ubiquitin-dependent degradation is involved in CXCL7 deficiency. Future studies in the CXCL7-null mouse model are, therefore, warranted to investigate the role of ubiquitination in the onset of OPLL. In conclusion, CXCL7 levels may be useful as a serum marker for the progression of OPLL. This study also suggests that increasing CXCL7 levels in patients can serve as an effective therapeutic strategy for the treatment of OPLL.

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.

Ubiquitin ligase UBE3C promotes melanoma progression by increasing epithelial-mesenchymal transition in melanoma cells.

Melanoma is the most aggressive type of skin cancer, exhibiting extensive local invasion and early distant metastasis. Aberrant expression of ubiquitin-protein ligase E3C (UBE3C) plays a key role in tumor development and progression. In the present study, we analyzed UBE3C expression in samples of cancerous and normal skin tissue. Levels of UBE3C expression were much higher in primary and metastatic melanoma tissues than in normal skin, cutaneous squamous cell carcinoma or basal cell carcinoma. Melanoma cells overexpressing UBE3C frequently exhibited a mesenchymal phenotype, including reduced expression of the epithelial marker E-cadherin and expression of the mesenchymal marker vimentin. Knockdown of UBE3C expression in melanoma cells significantly suppressed melanoma growth and progression. Furthermore, silencing UBE3C led to increased E-cadherin expression and decreased vimentin and Snail1 expression. Thus UBE3C promotes melanoma progression, possibly by inducing epithelial-mesenchymal transition in melanoma cells. Inhibiting UBE3C activity may suppress melanoma invasion and metastasis and may represent a targeted therapeutic approach.

Effect of UBE3C polymorphisms on intramuscular fat content and fatty acid composition in Duroc pigs.

Ubiquitin protein ligase E3C (UBE3C) is involved in the ubiquitin-proteasome pathway, and several ubiquitin protein ligases are important for fat deposition and lipid metabolism. The objective of this study was to analyze the association between a single nucleotide polymorphism (SNP) of the UBE3C gene with intramuscular fat (IMF) content and fatty acid (FA) composition in Duroc pigs. Four SNP markers (g.1586399A>G, g.1591358G>A, g.1600132G>C, and g.1600166G>A) of porcine UBE3C were genotyped using the polymerase chain reaction-restriction fragment length polymorphism method, and their associations with IMF content and FA composition were investigated in a commercial Duroc pig population. Two SNP markers (g.1586399A>G and g.1591358G>A) were segregated among the pigs. No UBE3C polymorphisms at g.1600132G>C or g.1600166G>A were observed. The UBE3C g.1586399A>G SNP was significantly associated with IMF content, while the UBE3C g.1591358G>A SNP was associated with palmitic, stearic, eicosenoic, and eicosadienoic acid levels, and saturated FA levels. These results suggest that polymorphisms in porcine UBE3C are correlated with IMF content and FA composition, and confirm the importance of porcine UBE3C as a candidate gene for fat deposition in pigs.

Ubiquitin-protein ligase E3C promotes glioma progression by mediating the ubiquitination and degrading of Annexin A7.

The ubiquitin-protein ligase E3C (UBE3C) belongs to the E3 ligase enzyme family and implicates in the ubiquitin-proteasome pathway, thus regulates physiological and cancer-related processes. Here, we investigated the expression and roles of UBE3C in glioma. We demonstrated that UBE3C was overexpressed in glioma tissues and cell lines. Inhibition of UBE3C expression in glioma cells significantly decreased cell migration and invasion in vitro. Mechanistically, we disclosed that UBE3C physically interacted with and ubiquitinated tumor suppressor gene annexin A7 (ANXA7), resulting in ubiquitination and degradation of ANXA7. Our results also revealed that increased UBE3C expression was accompanied by a reduction in ANXA7 protein expression in glioma tissues, but not ANXA7 mRNA. Importantly, the inhibition of ANXA7 expression in gliomas cells with UBE3C interference could rescue the cell invasion. Clinically, UBE3C overexpression significantly correlated with high-grade tumors (p < 0.05), poor overall survival, and early tumor recurrence. Thus, our data reveal that high UBE3C expression contributes to glioma progression by ubiquitination and degradation of ANXA7, and thus presents a novel and promising target for glioma therapy.

UBE3C genetic variations as potent markers of nasal polyps in Korean asthma patients

The human ubiquitin protein ligase E3C (UBE3C) regulates airway inflammatory responses and is hypothesized to be associated with the presence of nasal polyps in asthma-related diseases. A total of 24 UBE3C single-nucleotide polymorphisms (SNPs) were genotyped in a 467 Korean asthma cohort that was stratified into more homogenous phenotypes of 114 aspirin-exacerbated respiratory disease subgroup and 353 aspirin-tolerant asthma (ATA) subjects. Association analysis revealed that 16 UBE3C SNPs were significantly associated with presence of nasal polyps in the overall asthma group (P=0.0008 and P(corr)=0.01; odds ratio (OR)=0.60). The strength of association from 10 polymorphisms was increased in the ATA subgroup (P=0.0002 and P(corr)=0.003; OR=0.49). In addition, UBE3C_ht1 was found to be consistently associated with nasal polyps in the overall asthmatics group (P=0.006) and the ATA phenotype (P=0.002; P(corr)=0.02) via a codominant mechanism. Our findings provide evidence that variations in UBE3C are potent genetic markers of nasal polyps development in Korean asthmatics and may contribute novel insights into the clinical relevance and potential involvement of UBE3C in respiratory deficiencies.