Identification And Functional Studies Of BCCIP Interaction Proteins

BCCIP is a BRCA2and p21interaction protein. BCCIP play an important role in cellmitosis, cell cycle regulation, DNA homologous recombination repair, genome stability andthe development of nervous system. But the exact mechanism is not clear, to furtherinvestigate the function and mechanism of BCCIP, this project have found and identificatedproteins that can interact with BCCIP by using co-immunoprecipitation and yeast two hybridsystem.1. Based on the preliminary studies, we identified the interaction between PAK1IP1andBCCIPβ by co-immunoprecipitation that happened between extrinsic BCCIPβ with Flag-tagat the N-terminus and endogenous PAK1IP1in HT1080cells.2. I reconstructed the pOZ-FH-C1-BCCIP plasmid with a flag-tag at the C-terminus ofBCCIP, and the result that the extrinsic BCCIPβ-Flag produced by Hela cells transfectedwith this plasmid could pull down the endogenous protein PAK1IP1proved the interactionbetween PAK1IP1and BCCIPβ futher.3. After reconstructing a prokaryotic expression vector, pET-28b-PAK1IP1, Idiscovered that BCCIP marked by GST could be pulled down by PAK1IP1marked by His inco-immunoprecipitation which means that the interaction between PAK1IP1and BCCIPβmay be direct.4. I reconstructed full-length and7fragments plasmids of PAK1IP1marked by flag, andfound full-length PAK1IP1with Flag-tagged was able to pull down the endogenous BCCIPby co-immunoprecipitation. Then did the same experiments with7fragments plasmids ofPAK1IP1and identified the exact region of PAK1IP1required to interact with BCCIPβresides in amino acids330to392in the C-terminal.5. I constructed plasmids that express Flag tagged fragments of BCCIP and identifiedthat the region of BCCIP required to interact with PAK1IP1resides in amino acids1to167in the N-terminal by using co-immunoprecipitation.6. I designed shRNA of PAK1IP1, then constructed it into retroviral vector, pSilencer5.1-U6, after transfection, infected the HT1080cells with the supernatant, and then got themonoclone cell lines with PAK1IP1downregulated after selected by puromycin.7. After several experiments done with the cell lines mentioned above, we discoveredthat the down-regulation of PAK1IP1could induce the decreased growth of HT1080, but could not interfere with the expression of protein and the intracellular location of BCCIP.The γ-H2AX analysis and clone formation after radiation proved that the down-regulation ofPAK1IP1didn't result in the changes of the cellular radiosensitivity in HT1080cells.8. We identified3plasmids that interact with BCCIP by using yeast two hybrid system.The plasmids interacted with BCCIPα is proliferating cell nuclear antigen (PCNA) andFAM46A, the plasmid interacted with BCCIPβ is PCNA. In which, this is the first time thatFAM46A was discovered as an interaction protein to BCCIPα which may present a newaspect for the research in BCCIP.Conclusion:1. I discovered a new interaction protein PAK1IP1to BCCIPβ and interaction betweenthem may be direct; The region of PAK1IP1required to interact with BCCIPβ resides inamino acids330to392in the C-terminal; The region of BCCIPβ required to interact withPAK1IP1resides in amino acids1to167in the N-terminal.2. I reconstructed a model of down-regulation of PAK1IP1and found the knock downof PAK1IP1induced the decreased growth of HT1080, but didn't interfere with theexpression of protein and the intracellular location of BCCIP and unrelated to the cellularradiosensitivity.3. I identified PCNA and Fam46A interact with BCCIPα. This is the first time thatFAM46A was discovered as an interaction protein to BCCIPα which may present a newaspect for the research in BCCIP.