Studies On The Interaction Of ZCCHC10 Protein And P53 Protein

The tumor suppressor protein p53 functions as a transcriptional factor whose transcriptional activity is effected by the protein modification(e.g. ubiquitination and phosphorylation) and the interactions with other proteins. Recently, many proteins that interact with p53 were demonstrated to enhance or suppress the transcriptional activity of p53 by influencing the protein stability, the DNA binding activity or the subcellular localization of p53. ZCCHC10 was previously identified to be a possible p53-interacting protein by automated yeast two-hybrid screening. But the sequence of ZCCHC10 gene and the biological function generated by interaction with p53 have not been confirmed.We cloned the cDNA of ZCCHC10 and analyzed it by the bioinformatics. We found that the ZCCHC10 was located at chromosome 5q31.1. It was predicted to have two potential transcript variants. The longer transcript ZCCHC10-1 contains five extrons and four introns, encodes a protein with 192 amino acids. The start code locates in the first extron and the stop code exists in the fifth extron. The shorter ZCCHC 10-2 encodes a protein with 170 amino acids because of the loss of the second extron with 66 nucleotides. Both protein isoforms have a CCHC zinc finger.The rabbit polyclonal antibodies against ZCCHC10-1 and ZCCHC 10-2 were prepared to detect their protein expression in mammalian cell lines. Both ZCCHC10-1 and ZCCHC10-2 were highly expressed in normal cell line HBL100 and the p53-null breast cancer cell lines(MDA-MB453、MDA-MB231、T47D), but not in wild-type p53 tumor cell line MCF-7.We found that ZCCHC10-1 was located in the entire cell and the ZCCHC10-2 was only located in nucleus by the fluorescence microscopy. Both of them were co-localized with p53 in nucleus. Furthermore, we demonstrated the interaction between ZCCHC10 and p53 using co-immunoprecipitation method.We co-transfected the expression plasmids of ZCCHC10-1, ZCCHC10-2 and pp53-luc(containing the p53 response element) respectively and found that they can enhance the transcriptional activity of p53. In order to interpret the molecular mechanism of the influence of ZCCHC10 on the transcriptional activity of p53 protein, we analyzed the influence of ZCCHC10 on the stability of p53 protein. The results showed that with an increase of ZCCHC10-1 and ZCCHC10-2 protein, the protein level of p53 protein increased indicating that ZCCHC10 can increase the stability of p53 protein.In our study, We identified two isoforms of ZCCHC10, and found that ZCCHC10 can enhance the transcriptional activity of p53 by increasing the stability of p53 protein. These results offered a possible new pathway of p53 regulation.