Of Nyd-sp15 Subcellular Localization And Cell Proliferation

Purpose:Proteins of cytidine deaminase family have many functions, mostly of them connect with body immunity,virus infection,tumorigenesis and drug resistance closely. including body immunity,virus infection and tumorigenesis. NYD-SP15 is a new member of cytidine deaminase family we identified and the study of the function of it has creative significance.Earlier we confirmed the cytidine deaminise activity of NYD-SP15 in vitro and its relationship to spermatogenesis. In this article, we studied its activity style, expression in cells and tissues,cell location and infection to cell proliferation with a espection of giving a clear recgnation of NYD-SP15.Methods:We compared the sequences of different cytidine deaminase proteins with NYD-SP15 using bioinformatics softwares.Then expressed and purified NYD-SP15 protein and prepared antibody to NYD-SP15 which was used in detection of NYD-SP15 expression by immunohistochemistry in mouse testis and human breast carcinoma.We also identified the mRNA expression of NYD-SP15 in normal and tumor cells in RT-PCR.To study the location and nuclear location motifs of NYD-SP15 we constructed GFP-fusion NYD-SP15 and its mutants.In order to found homodimer function and interaction with importins of NYD-SP15 we used yeast two-hybrid system and GST-pulldown assays. We constructed wild type and site mutants NYD-SP15 which were transfected into RPE cells to compare there functions to cell proliferation by flow cytometry.Then we constructed and selected stable expressing NYD-SP15-HEK-TER and NYD-SP15-MCF7 cells using lentivirus vector and studied the activities of these cells by growth curve wound assay and flow cytometry.Results:NYD-SP15 is highly conserved with the other members of cytidine deaminase family,especially with CDD and DCTD and the similiarties were reflected in its conserved active sites HXE...PCXXC,NLS (Nuclear Localization Signal) NES(Nuclear Export Signal) and Leucine zipper motifs. NYD-SP15 were expressed strongly in mouse testis except interstitium, spermatospore and sperm but in all human breast carcinoma specimens.The mRNA of NYD-SP15 were expressed in many culture cell lines such as Hela,293T,MCF7,HEKTER.GFP-fusion NYD-SP15 locates in cytoplasm and/or nuclear and the percents are different in different cells, cytoplasm and nuclear in MCF7 and CHO, but promotely in nucleus in HEK-TER cells and cytoplasm in 293T.When the NLS motif was deleted NYD-SP15 was expressed in cytoplasm but GFP-NLS only show in nucleus.And then we confirmed the interaction of NYD-SP15 with importin a in yeast two-hybrid system. Both yeast two-hybrid system and GST-pulldown assays detected that NYD-SP15 could form homodimers.NYD-SP15 could inhibit G1 to S confert in cell circles of RPE cells and active sites mutants didn't has this function. And when we tested the stable transfected NYD-SP15 cells, we found that NYD-SP15 depressed the proliferation of HEK-TER and MCF7 and HEK-TER changed bigger and planer and there were many vesicles in the cytoplasm.Conclusion:NYD-SP15 has cytoplasm/nucleus shuttling and homo dimerization effects just like other cytidine deaminase family proteins.The active sites are necessary for NYD-SP15.There were some relationship of NYD-SP15 with tumorigenesis and spermatogenesis and NYD-SP15 is important for cell proliferation and regulation.