The ubiquitin-proteasome system plays an important role in protein degradation. The process of ubiquitination requires ubiquitin activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 to complete the coordination. Our previous studies have shown that HUWE1 (HECT, UBA and WWE domain containing 1), as an E3 ubiquitin ligase, can degrade epidermal growth factor receptor (EGFR) to inhibit renal tubulointerstitial fibrosis. However, E2 ubiquitin-conjugating enzymes binding to HUWE1 are still unclear. The aim of the present study was to identify E2 ubiquitin-conjugating enzymes of HUWE1. Real-time PCR was used to identify E2 ubiquitin-conjugating enzyme that may interact with HUWE1. The expression of E2 ubiquitin-conjugating enzyme was detected in kidney of unilateral ureteral obstruction (UUO) mice and HK-2 cells treated with transforming growth factor-β (TGF-β). The results showed that the expressions of E2 ubiquitin-conjugating enzyme UBE2Q2 were significantly down-regulated at both RNA and protein levels in UUO kidneys. The expression of UBE2Q2 was also down-regulated in HK-2 cells stimulated with TGF-β, which was consistent with the change in the expression of HUWE1. These findings indicated that UBE2Q2 expression was synergistic with HUWE1 in the injured kidney. Co-immunoprecipitation (Co-IP) experiments showed that HUWE1 interacted with UBE2Q2 in HK-2 cells. The co-localization of UBE2Q2 and HUWE1 was confirmed by cell immunofluorescence staining. After knocking down UBE2Q2 by siRNA, ubiquitin binding to HUWE1 and EGFR was decreased. In sum, our results demonstrated that UBE2Q2, ubiquitin-conjugating enzyme, works with HUWE1 to mediate ubiquitination and degradation of target protein in kidney.
Animals ; Cell Line ; Fibrosis ; Humans ; Kidney Diseases ; Mice ; Ubiquitin-Conjugating Enzymes/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; Ubiquitination

OBJECTIVETo clone and identify the gene encoding human ubiquitin binding enzyme 2 and study its expression pattern.

METHODSAccording to the sequence of human EST, which is highly homologous to the mouse ubiquitin binding/conjugating enzyme (E2), primers were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by bioinformatics technique and its expression pattern was studied by using multiple-tissue Northern blot.

RESULTSTwo cDNA clones encoding human ubiquitin conjugating enzyme have been isolated and identified. Both containing the ubiquitin conjugating domain, the 2 cDNA clones are 88% identical in amino acid sequences and splicing isoforms to each other only with an exon excised to form the short sequence. They belong to a highly conserved and widely expressed E2 enzyme family. Northern blot shows that they are expressed exclusively in adult human heart, placenta, and pancreas but no transcripts can be detected in brain, lung, liver, skeletal muscle or kidney.

CONCLUSIONSThe gene encoding human ubiquitin binding enzyme is expressed under temporal control. As a key enzyme in the degradation of proteins, ubiquitin conjugating enzymes play a central role in the expression regulation on the level of post-translation.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; genetics ; Female ; Humans ; Mice ; Molecular Sequence Data ; Myocardium ; metabolism ; Pancreas ; metabolism ; Placenta ; metabolism ; Rats ; Sequence Alignment ; Sequence Analysis, DNA ; Ubiquitin ; genetics ; Ubiquitin-Conjugating Enzymes ; biosynthesis ; chemistry ; genetics

Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.
Acanthamoeba castellanii/*growth & development/*metabolism ; Animals ; Gene Knockdown Techniques ; Lipid Metabolism ; Models, Molecular ; Molecular Sequence Data ; Protein Structure, Tertiary ; Protozoan Proteins/genetics ; RNA, Small Interfering/metabolism ; Rats ; Sequence Analysis, DNA ; Spores, Protozoan/*growth & development/*metabolism ; Ubiquitin-Conjugating Enzymes/genetics/*metabolism

Ubiquitin conjugating enzyme functions as the second enzyme required for protein ubiquitination and plays an important role in ubiquitin transferring and substrate specific recognition. UBE2W, a newly described member of E2 family, was formerly reported probably involving in phototransduction or retinal degeneration in Drosophila. In this study, we report that murine UBE2W harbors a typical UBC domain and is highly conserved in different vertebrate homologues. GST-tagged UBE2W was expressed in E. coli BL21 (DE3) and purified with GST affinity chromatography. Using this antigen, we generated and further separated rabbit polyclonal antibody of UBE2W, of which the activity and specificity were confirmed by immunoblotting of transiently expressed myc-UBE2W fusion protein. Wide expression of UBE2W was found in brain, muscle, heart, lung, liver, spleen, kidney and testis of mouse with the generated antibody, indicating the functional importance of this novel protein. Furthermore, the UBE2W highly expression was confined to the adult testis and was developmental stage-specific.
Animals ; Antibodies ; metabolism ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Escherichia coli ; genetics ; metabolism ; Male ; Mice ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Testis ; metabolism ; Tissue Distribution ; Ubiquitin-Conjugating Enzymes ; immunology ; metabolism

OBJECTIVETo investigate UbcH10 expression in hepatocellular carcinoma and explore its clinicopathological implications.

METHODSWe detected UbcH10 mRNA expression using RT-PCR in normal liver cell line, cancer cell lines, surgically removed hepatocellular carcinoma tissue and corresponding adjacent non-tumor tissue and evaluated the clinicopathological significance of UbcH10. Immunohistochemistry was performed to investigate UbcH10 protein expression in hepatocellular carcinoma tissue, the adjacent tissue, and normal liver tissue specimens.

RESULTSNormal liver cell line L02 showed significantly lower UbcH10 mRNA expression levels than the cancer cell lines BEL-7402, Hep3B, HepG2 and SMMC-7721 (P<0.05). UbcH10 mRNA expression was also was significantly higher in hepatocellular carcinoma tissues than in the corresponding non-tumor tissues (P<0.05). Clinicopathological evaluation suggested that UbcH10 expression was associated with tumor invasion of the portal vein, tumor size, TNM staging, and tumor differentiation (P<0.05). Immunohistochemistry identified stronger UbcH10 expression in hepatocellular carcinoma tissues than in the adjacent tissues and normal liver tissues (68.6%, 28.6%, and 26.7%, respectively).

CONCLUSIONUbcH10 is over-expressed in hepatocellular carcinoma and may serve as a novel biomarker as well as a therapeutic target of hepatocellular carcinoma.

Adult ; Aged ; Biomarkers, Tumor ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Hep G2 Cells ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Ubiquitin-Conjugating Enzymes ; genetics ; metabolism

Chemically defined medium is widely used for culturing mouse embryonic stem cells (mESCs), in which N2B27 works as a substitution for serum, and GSK3β and MEK inhibitors (2i) help to promote ground-state pluripotency. However, recent studies suggested that MEKi might cause irreversible defects that compromise the developmental potential of mESCs. Here, we demonstrated the deficient bone morphogenetic protein (BMP) signal in the chemically defined condition is one of the main causes for the impaired pluripotency. Mechanistically, activating the BMP signal pathway by BMP4 could safeguard the chromosomal integrity and proliferation capacity of mESCs through regulating downstream targets Ube2s and Chmp4b. More importantly, BMP4 promotes a distinct in vivo developmental potential and a long-term pluripotency preservation. Besides, the pluripotent improvements driven by BMP4 are superior to those by attenuating MEK suppression. Taken together, our study shows appropriate activation of BMP signal is essential for regulating functional pluripotency and reveals that BMP4 should be applied in the serum-free culture system.
Animals ; Bone Morphogenetic Protein 4/metabolism* ; Cell Differentiation ; Chromosomal Instability ; Endosomal Sorting Complexes Required for Transport ; Mice ; Mitogen-Activated Protein Kinase Kinases/metabolism* ; Mouse Embryonic Stem Cells/cytology* ; Pluripotent Stem Cells/cytology* ; Signal Transduction ; Ubiquitin-Conjugating Enzymes

hUBEW, a newly identified class I ubiquitin conjugating enzyme, probably plays an important role in tumorigenesis and DNA repair processes. RNA interference (RNAi) is a process in cells to degrade specific homologous mRNA by forming duplex RNA and has been developed into a powerful tool to study gene functions. In this study, the H1-U6 dual promoter RNAi plasmid was constructed and the target sequence for hUbe2w could be transcribed from both strands and form a double stranded RNA with two 5'Uridine overhangs, which closely resembles endogenous functional siRNA. The hUbe2w cDNA was amplified from reverse transcription of the 293FT total RNA by RT-PCR, and then cloned into the pGL3-Control, pCMV-myc and pDsRed-express-C1 plasmids respectively, which were selected as report vectors to detect the RNAi effects. The plasmids were co-transfected into HEK293FT cells, and then the luciferase activity and hUBE2W protein expression were measured respectively. The Resulted reduction of mRNA and protein level demonstrate that the targets of 125 and 259 could significantly inhibit the hUbe2w expression.
Base Sequence ; Humans ; Molecular Sequence Data ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; physiology ; RNA, Small Nuclear ; genetics ; Ubiquitin-Conjugating Enzymes ; genetics ; metabolism

BACKGROUND: Ubiquitin-conjugating enzyme E2C (UBE2C) has been shown to be associated with the occurrence of various cancers and involved in many tumorigenic processes. This study aimed to investigate the specific molecular mechanism through which UBE2C affects breast cancer (BC) proliferation. METHODS: BC-related datasets were screened according to filter criteria in the Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database. Then differentially expressed genes (DEGs) were identified using Venn diagram analysis. By using DEGs, we conducted the following analyses including Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI), and survival analysis, and then validated the function of the hub gene UBE2C using quantitative reverse transcription-polymerase chain reaction (RT-qPCR), cell counting kit-8 (CCK-8) assay, transwell assay, and Western blot assay. RESULTS: In total, 151 DEGs were identified from the GEO and TCGA databases. The results of GO analysis demonstrated that the DEGs were significantly enriched with mitotic nuclear division, lipid droplet, and organic acid-binding. KEGG analysis showed that the peroxisome proliferators-activated receptor (PPAR) signaling pathway, regulation of lipolysis in adipocytes, and proximal tubule bicarbonate reclamation were significantly enriched in the signal transduction pathway category. The top three hub genes that resulted from the PPI network were FOXM1, UBE2C, and CDKN3. The results of survival analysis showed a close relationship between UBE2C and BC. The results of CCK-8 and transwell assays suggested that the proliferation and invasion of UBE2C knockdown cells were significantly inhibited (P < 0.050). The results of Western blot assay showed that the level of phosphorylated phosphatase and tensin homology deleted on chromosome 10 (p-PTEN) was obviously increased (P < 0.050), whereas the levels of phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), and hypoxia-inducible factor-1 alpha (HIF-1α) were dramatically decreased (P < 0.050) in the UBE2C knockdown cell. CONCLUSION UBE2C can promote BC proliferation by activating the AKT/mTOR signaling pathway.
Biomarkers, Tumor ; Breast Neoplasms/pathology* ; Cell Proliferation/genetics* ; Computational Biology ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Signal Transduction/genetics* ; TOR Serine-Threonine Kinases/genetics* ; Ubiquitin-Conjugating Enzymes/metabolism*

OBJECTIVE: To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. METHODS: We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). RESULTS: In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis ( < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 ( < 0.01) and Bax ( < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 ( < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. CONCLUSIONS ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.
Acetates ; pharmacology ; Apoptosis ; drug effects ; genetics ; Autophagy ; drug effects ; Autophagy-Related Proteins ; metabolism ; Benzopyrans ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation ; Humans ; MCF-7 Cells ; Morpholines ; pharmacology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; metabolism ; Pyrans ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Triazines ; pharmacology ; Ubiquitin-Conjugating Enzymes ; metabolism

Ubiquitin-conjugating enzyme 9 (Ubc9), the sole conjugating enzyme for sumoylation, regulates protein function and plays an important role in tumorigenesis. Whether Ubc9 is involved in the chemoresistance of breast cancer remains unknown. In this study, we aimed to evaluate the contribution of Ubc9 in the chemoresistance of breast cancer. Immunohistochemistry (IHC) was used to examine the expression level of Ubc9. Chi-square test, Wilcoxon test, and one-way ANOVA were applied to analyze the relationship between Ubc9 expression, clinicopathologic features, and clinical response to neoadjuvant chemotherapy. The significance of variables for survival was analyzed by the Cox proportional hazards model in a multivariate analysis. Kaplan-Meier survival curves were plotted and log-rank test was performed. The proportion of Ubc9-positive cells was higher in invasive ductal carcinoma than in normal breast tissues [(48.48 ± 17.94)% vs. (5.82 ± 2.80)%, P < 0.001]. High Ubc9 expression was associated with poor differentiation (Χ² = 6.538, P = 0.038), larger tumor size (Χ² = 4.701, P = 0.030), advanced clinical stage (Χ² = 4.651, P = 0.031), lymph node metastasis (Χ² = 9.913, P = 0.010), basal-like phenotype (Χ² = 8.660, P = 0.034), and poor clinical response to neoadjuvant chemotherapy (Χ² = 11.09, P = 0.001). The expected 6-year cumulative disease-free survival rate was 87.32% in patients with low Ubc9 expression compared to 68.78% in those with high Ubc9 expression (Χ² = 4.289, P = 0.038). These data indicate that high Ubc9 expression correlates with poor response to chemotherapy and poor clinical prognosis.
Adult ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Breast Neoplasms ; drug therapy ; enzymology ; pathology ; surgery ; Carcinoma, Ductal, Breast ; drug therapy ; enzymology ; pathology ; surgery ; Cyclophosphamide ; therapeutic use ; Disease Progression ; Disease-Free Survival ; Drug Resistance, Neoplasm ; Epirubicin ; therapeutic use ; Female ; Fluorouracil ; therapeutic use ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Lymphatic Metastasis ; Mastectomy ; methods ; Middle Aged ; Neoadjuvant Therapy ; Neoplasm Staging ; Proportional Hazards Models ; Remission Induction ; Tumor Burden ; Ubiquitin-Conjugating Enzymes ; metabolism ; Up-Regulation