OBJECTIVEOsteosarcoma is the most common type of malignant bone tumor in children and adolescents. The role of E3 ligases in tumorigenesis is currently a focus in tumor research. In the present study, we investigated the role of the E3 ligase tripartite motif 21 (TRIM21) in osteosarcoma cell proliferation.

METHODS3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays were used to assess osteosarcoma cell viability. U2-OS cells stably carrying a recombinant lentivirus expressing tetracycline-regulated TRIM21 were screened. Co-immunoprecipitation was coupled with LCMS/MS analysis to identify novel interacting partners of TRIM21. Co-immunoprecipitation and bimolecular fluorescence complementation (BIFC) were performed to validate the interactions between TRIM21 and its novel partner YWHAZ. A TRIM21-ΔRING construct was generated to test the effects of TRIM21 ligase activity on YWHAZ.

RESULTSTRIM21 positively regulated osteosarcoma cell proliferation. Overexpression of TRIM21 enhanced osteosarcoma cell tolerance toward various stresses. YWHAZ protein was identified as a novel interacting partner of TRIM21 and its expression levels were negatively regulated by TRIM21. The RING domain of TRIM21 was required for TRIM21 negative regulation of YWHAZ expression. However, overexpression of YWHAZ did not affect positive regulation of osteosarcoma cell proliferation by TRIM21.

CONCLUSIONOur results further clarify the molecular mechanisms underlying the pathogenesis of osteosarcoma.

14-3-3 Proteins ; genetics ; metabolism ; Cell Proliferation ; genetics ; Humans ; Osteosarcoma ; genetics ; Ribonucleoproteins ; genetics ; metabolism ; Tumor Cells, Cultured

OBJECTIVE: To investigate the changes of GATA-1 protein expression during erythroid differentiation of K562 cells under hypoxia and how GATA-1 can regulate erythroid differentiation by up-regulating the expression of miR-451a and inhibiting the expression of 14-3-3ζ. METHODS: K562 cells were divided into 2 groups: the normoxia group and the hypoxia group, after the induction of hemin for 96 h, the positive cells rate of the benzidine staining, the mRNA expression of γ-globin and the expression of CD235a were detected, and the success of the model was verified. The changes of GATA-1 and miR-451a expression in the above-mentioned 2 groups, the changes of miR-451a expression after over-expressed GATA-1 were detected by Western blot and qRT-PCR. The cells in normoxic group and hypoxia group were divided into negative control group (NC group) and miR-451a over-expression group respectively, and the degree of erythroid differentiation in the four groups was judged according to the corresponding erythroid differentiation indexes, and the expression of 14-3-3ζ was detected by Western blot after over-expressed miR-451a. RESULTS: The positive cell rate of benzidine staining, mRNA expression of γ-globin and the expression of CD235a after 96 h induction by K562 cells under hypoxia were significantly higher than 0 h, suggesting that the erythroid differentiation model of K562 cells under hypoxia was replicated successfully. The expression levels of GATA-1 protein and miR-451a in the hypoxic group were significantly higher than that in the normoxic group (P<0.05). The expression level of miR-451a in hypoxia group was significantly higher than that in NC group after overexpressed GATA-1 (P<0.05). After over-expressed of miR-451a under hypoxia, the positive cell rate of benzidine staining, the mRNA expression level of γ-globin and the expression of CD235a were significantly higher than those in NC group (P<0.05). The expression level of 14-3-3ζ protein in miR-451a over-expressed group was lower than that in NC group under hypoxia (P<0.05). CONCLUSION Hypoxia can significantly increase the expression of GATA-1 protein, and the increase of GATA-1 expression can up-regulate the expression of miR-451a, thereby inhibiting the expression of 14-3-3ζ protein, which hinders the cell proliferation in erythroid differentiation model of K562 cells and plays an important role in promoting erythroid differentiation.
14-3-3 Proteins ; Cell Differentiation ; Erythroid Cells/metabolism* ; GATA1 Transcription Factor/metabolism* ; Humans ; Hypoxia ; K562 Cells ; MicroRNAs/genetics*

To investigate changes of 14-3-3beta from apoptosis induced by PrP106-126 polypeptide, HeLa cell was incubated with PrP106-126 for 4h or 8h. Nucleus changes and the expression of PARP were detected differently by Hoechst staining and Western blotting. Expressing of protein and mRNA from 14-3-3beta was determined by Western blotting and Real-time PCR. The results show that typical nucleus pyknosis and chip of apoptosis and degradation of PARP were induced by PrP106-126 peptide in HeLa cells. Degradation of 14-3-3beta appeared in apoptosis groups induced by PrP106-126 peptide. However, 14-3-3beta mRNA did not display any changes in apoptosis groups. This study indicated that degradation of antiapoptosis protein 143-3beta induced by PrP106-126 peptide may be one of pathogenesis mechanism of prion disease.
14-3-3 Proteins ; metabolism ; Apoptosis ; drug effects ; HeLa Cells ; Humans ; Peptide Fragments ; pharmacology ; Prions ; pharmacology ; Proteolysis ; drug effects

UNLABELLEDOBJECTIVE To study the potential interaction between PrP protein.

METHODSThe supernatant of health and scrapie-infected hamsters' brain homogenate was prepared, while various recombinant 14-3-3beta or PrP proteins were purified. The possible molecular interaction between 14-3-3beta proteins and PrP was tested by pull-down and immunoprecipitation assays.

RESULTSBoth native PrP(c) and its protease-resistant isoform (PrP(Sc)) formed complexes with 14-3-3beta. The full-length recombinant 14-3-3beta proteins interacted with PrP. The domain responsible for interacting 14-3-3beta was located at N-terminal of 14-3-3beta (residues 1 to 38).

CONCLUSIONThe studies of the association of PrP with 14-3-3beta may further provide insight into a potential role of 14-3-3beta in the biological function of PrP and the pathogenesis of prion disease.

14-3-3 Proteins ; metabolism ; Animals ; Binding Sites ; Brain Chemistry ; Cricetinae ; Endopeptidases ; metabolism ; PrPSc Proteins ; metabolism ; Prion Diseases ; pathology ; Prions ; metabolism ; Scrapie ; physiopathology

PRAS40, a proline-rich Akt substrate of 40 kD, is 14-3-3 binding protein. To study the interaction between PRAS40 and 14-3-3 isoforms, We constructed the expression vector pEG-PRAS40 (DNA-binding plasmid) and pJG-PRAS40 (transcriptional activity plasmid) in yeast using gateway cloning technology, then the plasmid of pEG-PRAS40/pJG-PRAS40 was co-transformed into yeast EGY48 strain with each pJG-14-3-3 /pEG-14-3-3 isoform plasmid. The co-transformation were tested by nutrition limitation growth analysis, beta-galactosidase color assay was used to study the interaction degree between PRAS40 and 14-3-3 isoforms. We confirmed successfully the construction of pJG-PRAS40 and pEG-PRAS40 with enzyme digestion. four 14-3-3 isoforms were found interacting with PRAS40 using yeast two-hybrid assay, the interaction degree of Epsilon was stronger than beta and zeta, tau was the weakest. Our result will be used to further study the biological function of PRAS40 and 14-3-3 as new drug target.
14-3-3 Proteins ; genetics ; metabolism ; Adaptor Proteins, Signal Transducing ; Humans ; Phosphoproteins ; genetics ; metabolism ; Protein Binding ; Protein Isoforms ; genetics ; metabolism ; Two-Hybrid System Techniques

The molecular interaction between PrP and 14-3-3 beta and the possible interactional domain between two proteins were studied by co-immunoprecipitation, pull down and FRET assays. The results showed that PrP protein could interact with 14-3-3 beta in vitro and in vivo. The domain which responded for the interaction was located at C-terminal of PrP (amino acid residues 106 to 126). This study of the interaction between PrP and 14-3-3 protein further provided the insight into the potential role of 14-3-3 in the biological function of PrP and the pathogenesis of prion disease.
14-3-3 Proteins ; metabolism ; Animals ; Brain ; metabolism ; Cricetinae ; Fluorescence Resonance Energy Transfer ; HeLa Cells ; Humans ; Immunoprecipitation ; Prions ; metabolism ; Protein Binding ; Rabbits

14-3-3 Proteins ; genetics ; metabolism ; Animals ; Brain ; pathology ; Cricetinae ; Internal Ribosome Entry Sites ; Open Reading Frames ; Prion Diseases ; genetics ; metabolism

Objective: To explore the molecular mechanisms of 14-3-3ζ in gemcitabine resistance in extranodal NK/T-cell lymphoma, nasal type (ENKTL) . Methods: The effects of cell proliferation and invasion were detected by cell counting kit-8 (CCK-8) assay and transwell assay. YTS cells were exposed to gradually increased concentrations of gemcitabine to establish gemcitabine-resistant YTS cells (YTS-gem) in vitro. 14-3-3ζ specific siRNA lentiviral vector was transfected into YTS and YTS-gem cells to downregulate 14-3-3ζ expression, and stable transfected cell clones were screened. The protein expression was determined by Western blot. Results: ①14-3-3ζ expression was significantly up-regulated in gemcitabine resistant YTS-gem cells, comparing with that of YTS cells (P<0.05) . ②The results of CCK-8 and transwell assay showed that downregulation of 14-3-3ζ significantly reduced the cell proliferation and invasion abilities (P<0.05) . ③Downregulation of 14-3-3ζ could restore gemcitabine sensitivity in gemcitabine resistant YTS-gem cells (P<0.05) . ④Western blotting results showed that knockdown of 14-3-3ζ significantly upregulated pro-apoptotic Bax, and downregulated anti-apoptotic Bcl-2, Caspase-3, cleaved caspase-3, Cyclin D1 in gemcitabine-resistant YTS-gem cells (P<0.05) . There was no significant difference in p53 ang P-gp expression levels. Conclusions: 14-3-3ζ was upregulated in gemcitabine resistant YTS cells. Overexpression of 14-3-3ζ promoted cell proliferation and enhanced cell migration. 14-3-3ζ contributed to gemcitabine resistance to ENKTL through anti-apoptosis.
14-3-3 Proteins/metabolism* ; Cell Line, Tumor ; Deoxycytidine/therapeutic use* ; Drug Resistance, Neoplasm ; Humans ; Lymphoma, Extranodal NK-T-Cell/drug therapy* ; Gemcitabine

14-3-3 proteins are important proteins in plants, as they regulate plant growth and development and the response to biotic or abiotic stresses. In this study, a 14-3-3 gene(GenBank accession: OM683281) was screened from the cDNA library of the medicinal species Salvia miltiorrhiza by yeast two-hybrid and cloned. The open reading frame(ORF) was 780 bp, encoding 259 amino a cids. Bioinformatics analysis predicted that the protein was a non-transmembrane protein with the molecular formula of C_(1287)H_(2046)N_(346)O_(422)S_9, relative molecular weight of 29.4 kDa, and no signal peptide. Homologous sequence alignment and phylogenetic tree analysis proved that the protein belonged to 14-3-3 family and had close genetic relationship with the 14-3-3 proteins from Arabidopsis thaliana, Oryza sativa, and Nicotiana tabacum. The 14-3-3 gene was ligated to the prokaryotic expression vector pGEX-4 T-1 and then transformed into Escherichia coli BL21 for the expression of recombinant protein. Real-time fluorescent quantitative PCR showed that the expression of this gene was different among roots, stems, leaves, and flowers of S. miltiorrhiza. To be specific, the highest expression was found in leaves, followed by stems, and the lowest expression was detected in flowers. S. miltiorrhiza plants were treated with 15% PEG(simulation of drought), and hormones salicylic acid, methyl jasmonate, and ethephon, respectively, and the expression of 14-3-3 gene peaked at the early stage of induction. Therefore, the gene can quickly respond to abiotic stresses such as drought and plant hormone treatments such as salicylic acid, jasmonic acid, and ethylene. This study lays the foundation for revealing the molecular mechanism of 14-3-3 protein regulating tanshinone biosynthesis and responding to biotic and abiotic stresses.
14-3-3 Proteins/metabolism* ; Amino Acid Sequence ; Cloning, Molecular ; Ethylenes/metabolism* ; Gene Expression Regulation, Plant ; Hormones/metabolism* ; Phylogeny ; Plant Growth Regulators/pharmacology* ; Plant Proteins/metabolism* ; Recombinant Proteins/genetics* ; Salicylic Acid/metabolism* ; Salvia miltiorrhiza/metabolism*

OBJECTIVETo investigate the expression of 14-3-3 sigma and heat shock protein 27 (HSP27) in colorectal carcinoma(CRC) tissue and its clinical significance.

METHODSThe expression of 14-3-3 sigma and HSP27 was detected by immunohistochemical staining in 50 pathologically verified CRC cases. The association of clinical data with 14-3-3 sigma and HSP27 expression was examined.

RESULTSThe positive expression rate of 14-3-3 sigma was 10% in normal control mucosa and 58% in CRC tissue (P<0.01). The positive expression rate of HSP27 was 16% in normal control mucosa and 54% in CRC tissue (P<0.01). No correlation between 14-3-3 sigma and HSP27 expression was found in CRC tissue (P>0.05). The expression of 14-3-3 sigma was associated with patient age, tumor diameter and lymph node metastasis (LNM) (P<0.05), but not with gender, tumor differentiation or serous membrane invasion (P>0.05). The expression of HSP27 was associated with LNM (P<0.05), but not with gender, age, differentiation, tumor diameter or serous membrane invasion (P>0.05).

CONCLUSIONThe abnormal expression of 14-3-3 sigma and HSP27 is significantly associated with LNM in CRC.

14-3-3 Proteins ; Adult ; Aged ; Biomarkers, Tumor ; metabolism ; Colorectal Neoplasms ; metabolism ; pathology ; Exonucleases ; metabolism ; Exoribonucleases ; Female ; HSP27 Heat-Shock Proteins ; metabolism ; Humans ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Young Adult