| Summary: | 碩士 === 國立陽明大學 === 藥理學研究所 === 95 === DEN2 core protein (DEN2C), the building block of the nucleocapsid, is detected not only in the cytoplasm but also in the nucleoli of infected cells. Currently, little is known about the detailed properties of DEN2C. In this study, the cellular distribution of DEN2C was observed by expressing fluorescent protein-fused DEN2C. The nuclear localization signal (NLS) motif of DEN2C was sufficient for nuclear localization and the truncation of its NLS motif didn’t abandon its cytoplasmic localization property. Without the homotypic interaction domain, DEN2C was located exclusively in the nucleolus. Comparing with living colors subcellular localization markers, DEN2C was partially located in the ER and Golgi apparatus, but the cytoplasmic localization of DEN2C was not disturbed by brefeldin A, a Golgi apparatus disrupting agent. Furthermore, the immunofluorescence staining indicated that DEN2C was colocalized with nucleolar phosphorprotein B23 that functions in ribosome biogenesis and cellular transcriptional regulation. The interaction of DEN2C with B23 was further confirmed by immunoprecipitation, and the NLS motif of DEN2C was essential for binding to B23. Considering that DEN2C is reported to inhibit the luciferase reporter activity of plasmid containing the enhancer elements of p53 in a dose-dependent manner. Upon siRNA-mediated reduction of intracellular B23, p53-driven reporter activity was also suppressed. Although both DEN2C and B23 can regulate p53 transcriptional activity, the mechanism of DEN2C inhibition was not regulated through the interaction of DEN2C with B23. Notably, aa 37 to 46 of DEN2C is involved in this inhibition. Taken together, these results suggest that DEN2C may have many effects on cellular gene regulation in DEN-infected cells.
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