Mouse TNF-α Inducible Protein TNFAIP1 Interacts With P50 And PCNA

Mouse TNFAIP1 protein is the first identified TNF- inducible protein. It is also termed B12 protein and its function has not been reported. We have found that Mouse TNFAIP1 is highly homologous to human PDIP1, suggesting that TNFAIP1 protein may be involved in the interactions with the subunit of DNA polymerase delta (p50) and proliferating cell nuclear antigen (PCNA). To reveal functions of mouse TNFAIP1, we isolated three genes (TNFAIP1, p50 and PCNA) from mouse liver, and cloned them in frame into a series of eukaryotic and protokaryonic expression vectors. The recombinant plasmids were transformed into E.coli. DH5a, and the positive clones were selected and the plasmid DNA was identified by restriction enzyme analysis and sequencing. After the transient transfection of HeLa cells with those recombinant plasmids, co-immunoprecipitations of mouse TNFAIPland p50, mouse TNFAIP1 and PCNA were performed and Western blot was used to detect the interactions between them. In addition, we have purified recombinant proteins with different tags expressed in E.coli. DE3, and used the pull-down experiment to directly monitor interactions between them.The TNFAIP1 gene is mapped to chromosome 10q25 cM, spansapproximately 12.9 kb, and is composed of seven exons. It encodes a BTB/POZ domain at the N-terminus and a PCNA binding motif (QTKV-EFP) at the C-terminus. The BTB / POZ domain and the PCNA binding motif are highly conserved during the evolution. We revealed the direct interaction not only between mouse TNFAIP1 and P50, but also between mouse TNFAIP1 and PCNA. These suggest that mouse TNFAIP1 may be involved in the process of DNA replication, which provide a basis for the functional studies of human TNFAIP1 gene.