Screening Of FKBP38 Interacting Proteins By Yeast Two-Hybrid System And Priliminary Study Of SQSTM1/p62 Functions

Proteins that are composed of more than one subunit are found in cells and many different classes of proteins were proved with the ability to specifically target and form non-covalent complexes with other proteins. Yeast two-hybrid technology is one of the most frequently used methods to study protein-protein interaction. FKBP38 (FK506-binding protein 38) a member of FKBP family, is a multifunctional protein, which regulate cell proliferation, apoptosis and other life processes through interaction with different protein, and may be associated with amino acids signal transduction in the cell. In this study, we screen the cDNA library of Inner Mongolia Cashmere goats (GFb) to get the unknown FKBP38 interacting proteins by the yeast two-hybrid technology and further validate the function of interacting proteins, which promote the study of the regulation mechanisms of FKBP38 and its interaction proteins in cell growth and metabolism.First, we get the FKBP38 interacting unknown proteins from the pGADT7-cDNA library of goat fetal fibroblast by yeast two-hybrid screening system, using pGBKT7-FKBP38 as a bait protein. Then the one-to-one transformation and screening studies were conducted to the positive clones. The cDNA fragments encoding the prey proteins were isolated from the positive clones, sequenced and analyzed. At last, We selected SQSTM1/p62 from FKBP38 interacting proteins as the next step research subject. Co-immunopricipitation (co-IP) was used to confirm the interaction of FKBP38 and SQSTM1/p62. We constructed SQSTM1/p62 expression vector and RNA interference vector to study the function of SQSTM1/p62 gene in GFb cell growth by MTT assay and flow cytometry analysis.The experimental results were as follows:1.7 positive clones were obtained after the Y2H yeast strain with pGBKT7-FKBP38 and Y187 yeast strain with pGADT7-cDNA were mated and selected by four rounds of deficiency medium and corresponding antibiotics screening; 2. The prey proteins interaction with FKBP38, which confirmed by one-to-one transformation and screening studies, were sequenced and analyzed by BLAST. The candidate genes were eukaryotic translation initiation factor 1 (EIF1),40S ribosomal protein S5-like (LOC102183449), CD81 molecule (CD81), actin gamma 2 (ACTG2), sequestosome 1 (SQSTM1) ArfGAP with coiled-coil, ankyrin repeat and PH domains 3 (ACAP3), PDZ and LIM domain 7 (PDLIM7); 3. Co-immunoprecipitation results showed that there is an interaction between FKBP38 and SQSTMl/p62; 4. CDS of SQSTM1/p62 in Inner Mongolia Cashmere goat was cloned, which length was 1147bp. The SQSTM1/p62 RNA interference vector pRNAT-U6.1-shSQSTM1/p62 and over expression vector pIRES2-EGFP-SQSTM1/p62 were successfully constructed; 5. The MTT assay results showed that the proliferation of SQSTM1/p62 RNA interference vector pRNAT-U6.1-shSQSTMl/p62 transfected cells were inhibited comparing with control group.6. Flow cytometry analysis results showed that the S phase cell ratio is 17.26% in SQSTM1/p62 RNA interference vector transfected group and S phase cell ratio is 36.72% in SQSTM1/p62 overexpression group, indicating the regulatory roles of SQSTM1/p62 gene in cell cycle.In conclusion, we obtained 7 FKBP38 interacting proteins using the yeast two-hybrid technology and further studies showed that SQSTM1/p62 play important roles in GFb cell proliferation and cell cycle. The studies of interaction of FKBP38 and SQSTM1/p62 promote us to further explore the molecular mechanisms of amino acid signal transduction in the future.