High Level of Ubiquitin Conjugate Enzyme E2O Indicates Poor Prognosis of Patients with Hepatocellular Carcinoma

Objective Ubiquitin conjugate enzyme E2O(UBE2O)is a ubiquitin-conjugating enzyme that has been reported to be involved in tumorigenesis.This study investigated the role of UBE2O in hepatocellular carcinoma(HCC).Methods The expression of UBE2O was detected using qRT-PCR,Western blotting,and immunohistochemical staining.Cell proliferation and Transwell assays were used to detect proliferation,migration,and invasion of HCC cells,respectively.Bioinformatic analysis was performed to analyze the relationship between UBE2O and the clinical features,prognosis,and immune cell infiltration of HCC.Results UBE2O was significantly over-expressed in HCC tissues.High expression of UBE2O was associated with poor tumor grade and poor prognosis.Functional experiments showed that down-regulation of UBE2O inhibited HCC cell proliferation,migration,and invasion.Co-expression gene analysis and gene set enrichment analysis showed that UBE2O was associated with protein hydrolysis,cell cycle,and cancer-related pathways in HCC.The results of immune analysis revealed that the expression of UBE2O was positively correlated with the immune infiltration and expression of immune-related chemokines of HCC.Conclusions UBE2O is significantly correlated with the prognosis of HCC and may be a valuable prognostic biomarker for HCC.

Cloning and Phylogenetic Analysis of Ubiquitinconjugating Enzyme Gene TaUBC4 in Different Wheat Cultivars

[Objective] This study aimed to clone ubiquitin-conjugating enzyme gene TaUBC4 from different wheat cultivars and thus analyze their phylogenetic relationship.[Method] The UBC4 coding sequences were cloned through reverse transcription PCR （RT-PCR） from 21 wheat varieties.After sequencing,the UBC4 sequence in wheat cultivar Zhongguochun （GenBank accession No：M28059） was selected as the reference gene,to analyze the mutation frequency and evolutionary distance in the CDSs and corresponding amino acid sequences of the different wheat cultivars.Moreover,the phylogenetic tree based on the amino acid sequences of these TaUBC4 genes were constructed,involving the homologous sequences of TaUBC4 in eight other monocots.[Result] TaUBC4 sequence was highly conserved because the similarity in DNA sequences of the wheat varieties was over 94％,while that in amino acid sequence was over 96％.And the amino acid sequence difference only can be seen at two sites among some varieties.Phylogenetic tree constructed revealed the evolutionary relationships among these wheat varieties.[Conclusion] This study reveals the polymorphism and evolutionary characteristics in the nucleotide and amino acid sequences in different wheat varieties,which lays foundation for investigating the evolution and biological function of TaUBC4 gene.In addition,the phylogenetic tree constructed provides theoretical references for the classification of the wheat varieties with complicated background.

CLONING AND CHARACTERIZATION OF HUMANUBIQUITIN BINDING ENZYME 2 cDNA

To clone and identify the gene encoding human ubiquitin binding enzym e 2 and study its expression pattern. Methods. According to the sequence of human EST, which is highly homologous to t he mouse ubiquitin binding/conjugating enzyme (E2), primers were synthesized to screen the human fetal brain cDNA library. The gene was analyzed by bioinformati cs technique and its expression pattern was studied by using multiple tissue No rthern blot. Results. Two cDNA clones encoding human ubiquitin conjugating enzyme have been i solated 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 th at they are expressed exclusively in adult human heart, placenta, and pancreas b ut no transcripts can be detected in brain, lung, liver, skeletal muscle or kidn ey. Conclusions. The gene encoding human ubiquitin binding enzyme is expressed under temporal control. As a key enzyme in the degradation of proteins, ubiquitin con jugating enzymes play a central role in the expression regulation on the level o f post translation.

BIR repeat-containing ubiquitin conjugating enzyme (BRUCE) regulation of β-catenin signaling in the progression of drug-induced hepatic fibrosis and carcinogenesis

BACKGROUND BIR repeat-containing ubiquitin conjugating enzyme(BRUCE)is a liver tumor suppressor,which is downregulated in a large number of patients with liver diseases.BRUCE facilitates DNA damage repair to protect the mouse liver against the hepatocarcinogen diethylnitrosamine(DEN)-dependent acute liver injury and carcinogenesis.While there exists an established pathologic connection between fibrosis and hepatocellular carcinoma(HCC),DEN exposure alone does not induce robust hepatic fibrosis.Further studies are warranted to identify new suppressive mechanisms contributing to DEN-induced fibrosis and HCC.AIM To investigate the suppressive mechanisms of BRUCE in hepatic fibrosis and HCC development.METHODS Male C57/BL6/J control mice[loxp/Loxp;albumin-cre(Alb-cre)-]and BRUCE Alb-Cre KO mice(loxp/Loxp;Alb-Cre+)were injected with a single dose of DEN at postnatal day 15 and sacrificed at different time points to examine liver disease progression.RESULTS By using a liver-specific BRUCE knockout(LKO)mouse model,we found that BRUCE deficiency,in conjunction with DEN exposure,induced hepatic fibrosis in both premalignant as well as malignant stages,thus recapitulating the chronic fibrosis background often observed in HCC patients.Activated in fibrosis and HCC,β-catenin activity depends on its stabilization and subsequent translocation to the nucleus.Interestingly,we observed that livers from BRUCE KO mice demonstrated an increased nuclear accumulation and elevated activity ofβ-catenin in the three stages of carcinogenesis:Pre-malignancy,tumor initiation,and HCC.This suggests that BRUCE negatively regulatesβ-catenin activity during liver disease progression.β-catenin can be activated by phosphorylation by protein kinases,such as protein kinase A(PKA),which phosphorylates it at Ser-675(pSer-675-β-catenin).Mechanistically,BRUCE and PKA were colocalized in the cytoplasm of hepatocytes where PKA activity is maintained at the basal level.However,in BRUCE deficient mouse livers or a human liver cancer cell line,both PKA activity and pSer-675-β-catenin levels were observed to be elevated.CONCLUSION Our data support a“BRUCE-PKA-β-catenin”signaling axis in the mouse liver.The BRUCE interaction with PKA in hepatocytes suppresses PKA-dependent phosphorylation and activation ofβ-catenin.This study implicates BRUCE as a novel negative regulator of both PKA andβ-catenin in chronic liver disease progression.Furthermore,BRUCE-liver specific KO mice serve as a promising model for understanding hepatic fibrosis and HCC in patients with aberrant activation of PKA andβ-catenin.

Freezing-directed construction of enzyme/nano interfaces:Reagentless conjugation,superior activity,and better stability

Immobilizing enzyme to nano interfaces has demonstrated to be a favorable strategy for prompting the industrialized application of enzyme.Despite tremendous endeavor has been devoted to using gold nanoparticles(AuNPs)as conjugation matrix due to its fascinating physico-chemical properties,maintaining enzymatic activity while circumventing cumbersome modification remains a formidable challenge.Herein,the freezing-directed conjugation of enzyme/nano interfaces was constructed without extra reagent.As the proof of concept,glucose oxidase(GOx)was chosen as model enzyme.The one-pot conjugation process can be facilely completed at−20°C under aqueous solution.Moreover,with the loading of GOx on AuNP at freezing,the enzyme exhibited superior catalytic activity and stability upon thermal and pH perturbation.The mechanism of boosted activity was then discussed in detail.It was found that higher loading density under freezing condition and more enzyme tending to bind AuNPs via Au-S bond were the main factors for the superior activity.More importantly,this methodology was universal and can also be applied to other enzyme which contains natural cysteine,such as horseradish peroxidase(HRP)and papain.This facile conjugation strategy accompanied by remarkable bioactivity expand the possibilities for enzymatic biosensing,microdevice and even drug delivery.

Development of a competitive format sorbent assay for the determination of parathion in water using molecular imprinted polymer as specific sorbent carrier

A rapid,simple,and reliable competitive molecular imprinted polymer sorbent assay（MIPSA） was developed and validated for measurement of parathion in water samples.This assay employed a molecular imprinted polymer（MIP） that was synthesized with non-covalent imprinting method as capture reagent and p-aminoparathion conjugate of horseradish peroxidase（para-HRP） as an enzyme label.The assay depended on a competitive binding reaction between the enzyme conjugate and analyte for the binding sites of the MIP.The optimized analysis conditions of 10 ng mL-1 para-HRP and 10 mg mL-1 polymer were found.The assay was acceptable to detect parathion in water samples under the optimized conditions,with a limit of detection of 50 ng mL-1.Mean analytical recoveries of added parathion in water samples ranged from 101.2%to 105%.The precision of the assay was satisfactory; relative standard deviation ranged from 4.3%to 6%.

Small Ubiquitin-Like Modifier Conjugating Enzyme with Active Site Mutation Acts as Dominant Negative Inhibitor of SUMO Conjugation in Arabidopsis

Small ubiquitin-like modifier （SUMO） conjugation affects a broad range of processes in plants, including growth, flower initiation, pathogen defense, and responses to abiotic stress. Here, we investigate in vivo and in vitro a SUMO conjugating enzyme with a Cys to Ser change in the active site, and show that it has a dominant negative effect. In planta expression significantly perturbs normal development, leading to growth retardation, early flowering and gene expression changes. We suggest that the mutant protein can serve as a probe to investigate sumoylation, also in plants for which poor genetic infrastructure precludes analysis via loss-of-function mutants.