Screening And Identification Of Proteins Interacting To Death Receptor 6

The tumor necrosis factor superfamily (TNFSF) members and their corresponding receptor superfamily (TNFRSF) members are the key inducers and regulators in the cellular responses including cellular apoptosis, differentiation and proliferation etc., which are highly related to various physiological and pathological processes. At present, 19 different ligands and their 29 corresponding receptors have been identified, respectively. Death receptor 6 (DR6) or TNFRSF21, is a novel characterized TNFRSF. It is a potent receptor in inducing cellular apoptosis, while its corresponding ligand is unknown up to data. The purpose of the study is to screen and identify the possible ligand(s) or proteins interacting to DR6. A two hybrid system of Stratagene BacterioMatch was applied on this study. Two interacting molecules, one is hepsin, belongs to serine protease family, another is maspin, a member of serine protease inhibitor (serpin) family, were identified with the system. Both of them were expressed in E. coli, and the recombinant fusion proteins were proved to bind and interact to DR6 by NH2-terminated magnetic bead analytical method. Furthermore, the bioinformatics software for screening and analyzing for anti-sense functional peptides for protein-protein interaction was compiled on the basis of the theory of "Proteomic Code". The structural requirements for hepsin-DR6 and maspin-DR6 interactions were analyzed by the software. These results strongly suggested that the members of serine protease family or serpin were possible ligands or related proteins for DR6. These works provided the fruitful information for further elucidation of the structure and functions of DR6 and its ligands.1. Application of the bacterial two-hybrid system for the validation of extracellular protein-protein interactionsTo explore the possibility and feasibility in the analyzing extracellular protein-protein interactions using the bacterial two-hybrid system, the interactions of TNF-related apoptosis-inducing ligand (TRAIL) and its three receptors, DR4, DR5 andosteoclastogenesis inhibitory factor/osteoprotegerin (OCIF/OPG) were identified in the system of Stratagene BacterioMatch. A fused expression plasmid was constructed between pTRG with tetracycline (T) resistance and TRAIL'S soluble extracellular domain as the "bait", while the fused expression plasmids constructed between pBT with chloramphenicol (C) resistance and extracellular domain of DR4, DR5 or OCIF/OPG, respectively, as the "target". All the recombinant pTRG and pBT was co-transformed into bacterioMatch two-hybrid system reporter strain E. coli XL 1 -Blue MRF with kanamycin (K) resistance. The protein-protein interactions were detected by its carbenicillin (C)-resistant property on CTCK plate containing 250-500 ug/ml of C (carbenicillin), 1 5 ug/ml of T (tetracyclin), 34 urn/ml of C (chloramphenicol) and 50 ug/ml of K, and validated by galactosidase activation on X-Gal-phenylethyl p-D-thiogalactoside (PETG) plate. Four groups, including that co-transformed by pTRG-TRAIL/pBT-DR4, pTRG-TRAIL/ pBT-DR5, controls (pBT/LGF2 and PTRG/Galll), and pTRG-TRAIL/pBT-OPQ grew as carbenicillin-resistant colonies on the plates. The pre-three groups showed higher resistance to carbenicillin (above 500 fig/ml) and the last one to lower (under 250 ug/ml). The positive colonies were further validated by p-galactosidase activation on X-Gal-PETG plates. The others, including pTRG-TRAIL/pBT-LGF2, pBT-DR4 or pBT-DR5 or pBT-OPG/pTRG-Gal 1 1 , or non-co-transformed plasmids themselves, were negative on carbenicillin or X-Gal-PETG plates.The results showed that the bacterial two-hybrid system is possible and efficacious in analyzing and identifying some extracellular protein-protein interactions, since the recognitions and interactions between TRAIL and their receptors have been proved by others chemical or biologic methods. The results showed also that the affinity between TRAIL and OPG was lower than that between TRAIL and DR4 or DR5. 2. Screening the ligand candidate interacting to DR6 with the bac...