Objective: To explore the key deubiquitinating enzymes that maintain the stemness of liver cancer stem cells and provide new ideas for targeted liver cancer therapy. Methods: The high-throughput CRISPR screening technology was used to screen the deubiquitinating enzymes that maintain the stemness of liver cancer stem cells. RT-qPCR and Western blot were used to analyze gene expression levels. Stemness of liver cancer cells was detected by spheroid-formation and soft agar colony formation assays. Tumor growth in nude mice was detected by subcutaneous tumor-bearing experiments. Bioinformatics and clinical samples were examined for the clinical significance of target genes. Results: MINDY1 was highly expressed in liver cancer stem cells. The expression of stem markers, the self-renewal ability of cells, and the growth of transplanted tumors were significantly reduced and inhibited after knocking out MINDY1, and its mechanism of action may be related to the regulation of the Wnt signaling pathway. The expression level of MINDY1 was higher in liver cancer tissues than that in adjacent tumors, which was closely related to tumor progression, and its high expression was an independent risk factor for a poor prognosis of liver cancer. Conclusion: The deubiquitinating enzyme MINDY1 promotes stemness in liver cancer cells and is one of the independent predictors of poor prognosis in liver cancer.
Animals ; Mice ; Cell Line, Tumor ; Mice, Nude ; Liver Neoplasms/pathology* ; Prognosis ; Deubiquitinating Enzymes/metabolism* ; Neoplastic Stem Cells/pathology* ; Gene Expression Regulation, Neoplastic

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

Enzyme separation, purification, immobilization, and catalytic performance improvement have been the research hotspots and frontiers as well as the challenges in the field of biocatalysis. Thus, the development of novel methods for enzyme purification, immobilization, and improvement of their catalytic performance and storage are of great significance. Herein, ferritin was fused with the lichenase gene to achieve the purpose. The results showed that the fused gene was highly expressed in the cells of host strains, and that the resulted fusion proteins could self-aggregate into carrier-free active immobilized enzymes in vivo. Through low-speed centrifugation, the purity of the enzymes was up to > 90%, and the activity recovery was 61.1%. The activity of the enzymes after storage for 608 h was higher than the initial activity. After being used for 10 cycles, it still maintained 50.0% of the original activity. The insoluble active lichenase aggregates could spontaneously dissolve back into the buffer and formed the soluble polymeric lichenases with the diameter of about 12 nm. The specific activity of them was 12.09 times that of the free lichenase, while the catalytic efficiency was 7.11 times and the half-life at 50 ℃ was improved 11.09 folds. The results prove that the ferritin can be a versatile tag to trigger target enzyme self-aggregation and oligomerization in vivo, which can simplify the preparation of the target enzymes, improve their catalysis performance, and facilitate their storage.
Biocatalysis ; Enzymes, Immobilized/metabolism* ; Ferritins/metabolism* ; Glycoside Hydrolases/metabolism*

Ubiquitination is one of the reversible protein post-translational modifications, in which ubiquitin molecules bind to the target protein in a cascade reaction of ubiquitin activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases. The deubiquitinating enzymes (DUBs) remove ubiquitin residues from the substrates, which play key roles in the formation of mature ubiquitin, the removal and trimming of ubiquitin chains, as well as the recycling of free ubiquitin chains. Ubp14, a member of the ubiquitin specific proteases family in Saccharomyces cerevisiae, is mainly responsible for the recycling of intracellular free ubiquitin chains. To investigate its global biological function, a ubp14∆ mutant was constructed by homologous recombination technique. The growth rate of ubp14∆ mutant was lower than that of the wild-type (WT) strain. Using stable isotope labeling by amino acids in cell culture (SILAC) combined with deep coverage proteomics analysis, the differentially expressed proteins of ubp14∆ mutant relative to the wild-type strain were systematically analyzed. A total of 3 685 proteins were identified in this study, and 109 differentially expressed proteins were filtered out by statistical analysis. Gene ontology analysis found that differentially expressed proteins caused by Ubp14 loss were mainly involved in amino acid metabolism, REDOX, heat shock stress and etc, which shed light on the broad biological function of this DUB. This study provides highly reliable proteomic data for further exploring the biological functions of the deubiquitination enzyme Ubp14, and further understanding the relationship between the free ubiquitin homeostasis and biological process regulation.
Saccharomyces cerevisiae/metabolism* ; Proteomics ; Endopeptidases/metabolism* ; Ubiquitin/metabolism* ; Ubiquitination ; Proteins/metabolism* ; Deubiquitinating Enzymes/metabolism* ; Biological Phenomena

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

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca₃(PO₄)₂ hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca₃(PO₄)₂ hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca₃(PO₄)₂ hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca₃(PO₄)₂ hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4 ℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The K_m of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the k_cat/K_m was (13 151.53± 299.19) L/(mmol·s). The K_m of the KatA/Ca₃(PO₄)₂ hybrid nanoflowers was (32.75±2.96) mmol/L, and the k_cat/K_m was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca₃(PO₄)₂ hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca₃(PO₄)₂ hybrid nanoflowers using Ca²⁺ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.
Catalase ; Nanostructures/chemistry* ; Hydrogen Peroxide/metabolism* ; Enzymes, Immobilized/chemistry* ; Catalysis

The development of biotechnology and the in-depth research on disease mechanisms have led to increased application of enzymes in the treatment of diseases. In addition, enzymes have shown great potential in drug manufacturing, particularly in production of non-natural organic compounds, due to the advantages of mild reaction conditions, high catalytic efficiency, high specificity, high selectivity and few side reactions. Moreover, the application of genetic engineering, chemical modification of enzymes and immobilization technologies have further improved the function of enzymes. This review summarized the advances of using enzymes as drugs for disease treatment or as catalysts for drug manufacturing, followed by discussing challenges, potential solutions and future perspectives on the application of enzymes in the medical and pharmaceutical field.
Biocatalysis ; Biotechnology ; Catalysis ; Drug Compounding ; Enzymes/metabolism*

A stable Zr-based metal-organic framework (MOF, UiO-66-NH2) synthesized via micro-water solvothermal method was used to immobilize amidase by using the glutaraldehyde crosslinking method. The effect of immoblization conditions on enzyme immoblization efficiency was studied. An activity recovery rate of 86.4% and an enzyme loading of 115.3 mg/g were achieved under the optimal conditions: glutaraldehyde concentration of 1.0%, cross-linking time of 180 min, and the weight ratio of MOF to enzyme of 8:1. The optimal temperature and optimal pH of the immobilized amidase were determined to be 40 °C and 9.0, respectively, and the Km, Vmax and kcat of the immoblized amidase were 58.32 mmol/L, 16.23 μmol/(min·mg), and 1 670 s⁻¹, respectively. The immobilized enzyme was used for (S)-4-fluorophenylglycine synthesis and the optimal reaction conditions were 300 mmol/L of N-phenylacetyl-4-fluorophenylglycine, 10 g/L of immobilized enzyme loading, and reacting for 180 min at pH 9.0 and 40 °C. A conversion rate of 49.9% was achieved under the optimal conditions, and the conversion rate can be increased to 99.9% under the conditions of enantiomeric excess. The immobilized enzyme can be repeatedly used, 95.8% of its original activity can be retained after 20 cycles.
Amidohydrolases ; Enzyme Stability ; Enzymes, Immobilized/metabolism* ; Glycine/analogs & derivatives* ; Hydrogen-Ion Concentration ; Metal-Organic Frameworks ; Temperature

To enhance recombinant protein production by CHO cells, We compared the impact of overexpression of metabolic enzymes, namely pyruvate carboxylase 2 (PYC2), malate dehydrogenase Ⅱ (MDH2), alanine aminotransferase Ⅰ (ALT1), ornithine transcarbamylase (OTC), carbamoyl phosphate synthetase Ⅰ (CPSⅠ), and metabolism related proteins, namely taurine transporter (TAUT) and Vitreoscilla hemoglobin (VHb), on transient expression of anti-hLAG3 by ExpiCHO-S. Overexpression of these 7 proteins could differentially enhance antibody production. OTC, CPSI, MDH2, and PYC2 overexpression could improve antibody titer by 29.2%, 27.6%, 24.1%, and 20.3%, respectively. Specifically, OTC and MDH2 could obviously improve early-stage antibody production rate and the culture period was shortened by 4 days compared with that of the control. In addition, OTC and MDH2 had little impact on the affinity of anti-hLAG3. In most cases, overexpression of these proteins had little impact on the cell growth of ExpiCHO-S. MDH2 and ALT1 overexpression in H293T cells could also improve antibody production. Overall, overexpression of enzymes involved in cellular metabolism is an effective tool to improve antibody production in transient expression system.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Enzymes/metabolism* ; Recombinant Proteins/genetics*

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*