OBJECTIVES: To investigate the role of nucleolar pre-rRNA processing protein NIP7 (NIP7) in maintaining the malignant phenotype of anaplastic thyroid cancer (ATC) and its molecular mechanisms. METHODS: NIP7 expression in ATC tissues and its gene knock-out effects in ATC cells were analyzed using gene expression microarray (GSE33630), proteome database (IPX0008941000) and the Dependency Map database, respectively. Expression and localization of NIP7 in normal thyroid cells, papillary thyroid cancer cells, and ATC cells were detected by Western blotting. Small interfering RNA (siRNA) was transfected into ATC cells, and the knockdown efficiency of NIP7 was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. Cell proliferation was assessed by CCK-8 assay, colony formation was evaluated by colony formation assay, and tumor growth was assessed by xenograft tumor model in nude mice. SUnSET (surface sensing of translation) assay combined with co-immunoprecipitation were employed to evaluate the effect of NIP7 silencing on ubiquitin-conjugating enzyme E2 C (UBE2C) translation. Finally, gene set enrichment analysis was used to identify shared pathways of NIP7 and UBE2C, which were validated by qRT-PCR. RESULTS: Compared with normal tissues and papillary thyroid cancer, NIP7 was significantly upregulated in ATC tissues, and had a gene knock-out fitness effect on different ATC cell lines. The relative protein levels of NIP7 in ATC cells were significantly higher than those in normal thyroid follicular cells, and the protein was mainly expressed in the nucleus. NIP7 silencing significantly inhibited cell proliferation and reduced colony formation. Xenograft tumor model showed that NIP7 knockdown significantly slowed down the growth of ATC xenograft, and the tumor volume and weight were significantly lower than those in the control group (all P<0.05). NIP7 silencing downregulated the protein level of UBE2C, but did not affect the expression of UBE2C mRNA. Compared to the control group, UBE2C silencing significantly inhibited ATC cells proliferation (P<0.01) and colony formation (P<0.05). UBE2C overexpression reversed the proliferation-inhibitory effect induced by NIP7 silencing (P<0.01). Gene set enrichment analysis indicated that NIP7 and UBE2C were both involved in DNA replication. NIP7 or UBE2C silencing could significantly downregulate the expression levels of DNA polymerase epsilon, catalytic subunit 2 and replication factor C4 in DNA replication pathway. CONCLUSIONS NIP7 promotes ATC tumor growth by upregulating UBE2C to mediate DNA replication.
Humans ; Ubiquitin-Conjugating Enzymes/genetics* ; Thyroid Neoplasms/genetics* ; Thyroid Carcinoma, Anaplastic/genetics* ; Animals ; Mice, Nude ; Mice ; Cell Line, Tumor ; Cell Proliferation ; Up-Regulation ; RNA, Small Interfering/genetics* ; Nuclear Proteins/metabolism* ; Gene Expression Regulation, Neoplastic

As the chip of synthetic biology, enzymes play a vital role in the bio-manufacturing industry. The development of diverse functional enzymes can provide a rich toolbox for the development of synthetic biology. This article reports the construction of an artificial enzyme with the introduction of a non-natural cofactor. By introducing the 4-dimethylaminopyridine (DMAP) cofactor into the optimal protein skeleton via covalent bonds based on a click-chemistry strategy, we successfully constructed a novel artificial enzyme with the DMAP cofactor as the catalytic center. The artificial enzyme successfully catalyzed an unnatural asymmetric Morita-Baylis- Hillman (MBH) reaction between cycloketenone and p-nitrobenzaldehyde, with a conversion rate of 90% and enantioselectivity (e.e.) of 38%. This study not only provides an effective strategy for the design of new artificial enzymes but also establishes a theoretical basis for the development of unnatural biocatalytic MBH reactions.
Biocatalysis ; 4-Aminopyridine/chemistry* ; Enzymes/metabolism* ; Coenzymes/chemistry* ; Benzaldehydes/chemistry* ; Protein Engineering/methods* ; Click Chemistry

Penicillin G acylases (PGAs) are industrially important enzymes primarily used for the synthesis of first- and second-generation cephalosporins or penicillins. This study aims to establish a high-efficiency biosynthetic system for cefradine on the purpose of significantly enhancing its catalytic efficiency in cefradine synthesis and developing its potentials for industrial application. In this study, we identified and engineered penicillin G acylase and obtained a highly active mutant KsPGA M7(M168F/F313G) for the synthesis of cefradine. The mutant achieved a conversion rate over 95% in the scaled-up reaction. To validate its industrial applicability, we immobilized both the wild-type and mutant enzymes and applied them in continuous flow reactions, which achieved a space-time yield of 2 800 g/(L·d). This study lays a foundation for the future applications of penicillin G acylases in the industrial synthesis of cefradine.
Penicillin Amidase/biosynthesis* ; Protein Engineering/methods* ; Cephradine/metabolism* ; Escherichia coli/metabolism* ; Enzymes, Immobilized/metabolism* ; Recombinant Proteins/biosynthesis*

The structures and activities of enzymes are influenced by pH of the environment. Understanding and distinguishing the adaptation mechanisms of enzymes to extreme pH values is of great significance for elucidating the molecular mechanisms and promoting the industrial applications of enzymes. In this study, the ESM-2 protein language model was used to encode the secreted microbial proteins with the optimal performance above pH 9 and below pH 5, which yielded 47 725 high-pH protein sequences and 66 079 low-pH protein sequences, respectively. A deep learning model was constructed to identify protein acid-base tolerance based on amino acid sequences. The model showcased significantly higher accuracy than other methods, with the overall accuracy of 94.8%, precision of 91.8%, and a recall rate of 93.4% on the test set. Furthermore, we built a website (https://enzymepred.biodesign.ac.cn), which enabled users to predict the acid-base tolerance by submitting the protein sequences of enzymes. This study has accelerated the application of enzymes in various fields, including biotechnology, pharmaceuticals, and chemicals. It provides a powerful tool for the rapid screening and optimization of industrial enzymes.
Deep Learning ; Hydrogen-Ion Concentration ; Amino Acid Sequence ; Enzymes/metabolism* ; Sequence Analysis, Protein ; Proteins/metabolism* ; Bacterial Proteins/metabolism*

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

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

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

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*