NYD-SP15 subcellular localization and cell proliferation
|School||Nanjing Medical University|
|Keywords||Cytidine deaminase NYD-SP15 Dimerization Subcellular localization Cell proliferation|
Background Objective: cytidine deaminase family protein function is complex and diverse, many members of the immune, viral infections, tumors and tumor resistance are closely related. NYD-SP15, we found that the new members of the family, its function has groundbreaking significance. In this study, the expression of NYD-SP15 biological activity, subcellular localization, cell and tissue distribution, and impact on cell proliferation studies, preliminary clarify NYD-SP15 gene function. Methods: NYD-SP15 by bioinformatics software analysis and comparison to its similarity to the sequence of the same family of proteins, analysis of functional domains, suggesting that its function; prokaryotic expression and purification of NYD-SP15 protein polyclonal antibody by immunohistochemistry analysis NYD-SP15 NYD-SP15 by RT-PCR analysis in testicular, breast carcinoma and distribution; through to build NYD-SP15 their nuclear localization signal mutant green fluorescent fusion protein expression in some normal cells and tumor cells; tablets, its intracellular localization and possible nuclear localization sequence; into-importins the interactions of nucleoprotein receptor by the yeast two-hybrid and GST-pulldown test analysis NYD-SP15 homologous dimerization and NYD-SP15 ; build NYD-SP15 wild type and mutations to the active site of the plasmid was determined by flow cytometry after transfection of RPE cell cycle research NYD-SP15 active sites; slow build HEK-TER and MCF7 cells NYD-SP15 The virus stably expressing cell lines, crossed experiments and cell growth curve analysis NYD-SP15 role in cell migration and cell proliferation, and cell cycle was detected by flow cytometry. Results: NYD-SP15 highly conserved in different species, cytidine off the ammonia enzyme domain the DCTD most similar of all cytidine deaminase highly conserved HXE ... PCXXC sequence and there into nuclear signal sequence, a nuclear export signal sequence and the leucine zipper sequences. NYD-SP15 is highly expressed in the mature mouse testis interstitial spermatocytes and sperm low expression or no expression; highly expressed in breast cancer tissue microarray specimens. RT-PCR NYD-SP15 expression in Hela, 293T, MCF7, HEKTER cultured cells. NYD-SP15 can be positioned in the cell nucleus and / or cytoplasm, the proportion is different in different cells, MCF7 and CHO in the nucleus and cytoplasm are distributed, HEK-TER nucleus mainly 293T cytoplasmic mainly. NYD-SP15 deleting the nuclear localization signal is located in the cytoplasm, and the nuclear localization signal GFP fusion protein is only expressed in the nucleus. NYD-SP15 existence confirmed by nuclear localization signal mutation double-digit point into a nuclear signal sequence of the yeast two-hybrid confirmed NYD-SP15 interaction into the nucleoprotein receptor importin a. Yeast two-hybrid and GST-pulldown confirmed NYD-SP15 can form homodimers in vitro. The cell cycle of wild-type NYD-SP15 RPE cells from G1 to S phase arrest in front off the ammonia enzyme active site domain mutations have a significant impact on this role. Stably transfected cell lines and growth curve, crossed experimental determination also showed a significant inhibition of HEK-TER and MCF7 cell growth and cell cycle. In addition, stable cell lines transfected HEK-TER morphology also occurred changes in cell flat and larger volume than the control, there are many different sizes of vacuoles in the cytoplasm. Conclusion: NYD-SP15 presence of their homologous protein similar pulp / nuclear shuttle regulation mechanism and the active site and dimerization effect; functionality of the the conservative active sites on NYD-SP15 is necessary; NYD-SP15 spermatogenesis and tumor there is a certain relationship occurs; affect cell growth and regulation.