| FAM76B gene is also known as MGC33371,which is located on chromosome 11q21 with a full-length 1020bp cDNA,encoding 339 amino acids,in its 151-160aa is a poly-serine region,and its 167-187aa is a His rich region.So far,the specific biological function of FAM76B is poorly understood.The previous study has reported that FAM76B gene interacts with KRTAP4-12,which is a member of the KAP protein family and is associated with the formation of hair fiber structure.Our lab previously study proofed that PGRN interacts with FAM76B.Recently research shown that PGRN is closely related with inflammation,tissue repair and embryonic development.Meanwhile,the function of PGRN is also associated with tumor angiogenesis,proliferation.In addition,PGRN mutations lead to haploinsufficiency(Haploinsufficiency)which will cause ubiquitin-positive inclusions of frontotemporal lobar degeneration(FTLD-U).As a PGRN interacting protein,the research of FAM76B will have an practical meaning on our understanding of the function of PGRN.Aim to get a further knowledge about the function of FAM76B,we used yeast two-hybrid system to select the FAM76B interacting protein.Interestingly,expect PGRN was the most frequently detected protein and MDFIC(MyoD family inhibitor domain containing protein,MDFIC)was also detected in some cases.MDFIC is a key target protein in our project,as MDFIC interacts with PGRN and with other important proteins,such as HIV-1 Tat,cyclin T1 and so on.Then,we used co-immunoprecipitation(CoIP),GST-pull down technic and Confocal laser microscopy to validate the interaction between FAM76B and MDFIC.Last,we studied the biological function of the protein interaction between FAM76B and MDFIC.The aims of the research project are as follows:1.The screening of FAM76B interacting proteins using the yeast two-hybrid system.PGBKT7/FAM76B vector was constructed and used as a bait to screen FAM76B interacting proteins from human leukocyte cDNA library using the two-hybrid assay.Through sequencing and identification of screened genes we identified that PGRN was the most frequently detected protein and MDFIC.2.Validation of the interaction between FAM76B and MDFIC.We cloned full length MDFIC molecular from human cDNA library by PCR technic,and then cloned it into yeast vector.Moreover,we validated the molecular interaction between FAM76B and MDFIC by yeast two-hybrid system.By constructing the deletions of MDFIC and cloned them to yeast vector,using yeast two-hybrid system we studied the interaction domains of the two proteins.The result showed that the interaction domain of FAM76B located on its His-repeat tract,and the precise interaction region of MDFIC resides within I-mfa domain.Next,the interaction between FAM76B and MDFIC was convinced by the CoIP in vivo and GST pull-down assay in vitro.Finally,the results of confocal laser microscopy showed that co-expression of FAM76B with MDFIC in the CHO cells,which promotes a translocation of FAM76B from the nuclei into the cytoplasm,and was colocalized with MDFIC there.3.Study on the biological function of FAM76B and MDFICFirst,we found endogenous expression of MDFIC in Hela cells,U20S cells and HepG2 cells using real-time PCR assay.It is reported that MDFIC interact with Axinl complex,and speculate MDFIC binding with Axinl may inhibit other protein binding to it.According to the report,we over expressed Axinl in HepG2 cells to suppress cell proliferation and analyzed by MTT assay.Then,the proliferation of overexpressed MDFIC or FAM76B in HepG2 cells was analyzed by MTT assay.We found that co-suppression effect between Axinl and MDFICp40,which presents a more significant suppression effect on the cell proliferation.In summary,some results obtained from the project as follows:(1)a novel FAM76B interacting protein MDFIC was identified by the yeast two-hybrid system.(2)The direct interaction between FAM76B and MDFIC was validated by the co-immunoprecipitated,GST pull-down assay and confocal laser microscopy.(3)By using gene deletion technique and the yeast two-hybrid system,the precise domain of FAM76B interacting with MDFIC was found to reside within the His-repeat tract,and the precise interaction region of MDFIC resides within I-mfa domain.(4)Real-time PCR assay analyzed endogenous expression of MDFIC in Hela,U20S,HepG2 and MCF-7 cell lines.(5)Over expression of Axinl and MDFICp40 in HepG2 cell line,cell proliferation was more significantly suppressed than single gene. |
