Construction Of Yeast Conformation-constrained Random Peptide Library And Screening For Dissociative Peptides Of BAFF-R/TRAF3 Complex

BAFF (B-cell-activating factor, also called BLyS, TALL-1, THANK, zTNF4 and TNFSF-13B), was discovered in 1999 as a member of the TNF superfamily. It can enhance B cell survival and maturation, and plays an important role in immune response, but excess production may cause multiple autoimmune diseases. BAFF binds to three receptors: BCMA, TACI, BAFF-R/BR3. BAFF/BAFF-R signaling plays critical roles in regulation of B cell function and autoimmune disease. TNFR-associated factor 3 (TRAF3) as a cytoplasmic protein physically binds to the cytoplasmic domain of BAFF-R. Inhibiting the interaction between BAFF-R and TRAF3 helps to block BAFF-R-mediated signaling pathway and to treat autoimmune disease. In our study, the peptides promoting dissociation of BAFF-R and TRAF3 were screened out of a yeast conformation-constrained random peptide library, and provided a new target for the treatment of autoimmune disease. This study consists of the following three parts:1. BAFF-R cytoplasmic domain and TRAF3-C (amino acids 258-568) were amplified by RT-PCR from B lymphoma cells (Raji). The recombinant plasmids pBD-BAFF-R and pAD-TRAF3-C were introduced into yeast cell to identify the interaction between BAFF-R and TRAF3-C. The recombinant plasmids pM-BAFF-R and pVpl6-TRAF3-C were transfected into COS7 cells and their interactions were identified. A series of deletion mutants of TRAF3 were made, their interaction with BAFF-R was tested in yeast. Our results showed that three parts of TRAF3 (amino acid residues 328-400, 428-463, 543-560) participate in the binding of BAFF-R.