Establishment Of An Inhibitor Screening System For The Interaction Between REG Gamma And Alpha 7 By Using Luciferase Fragment Complementation Technique

The Proteasome plays a very important role in maintaining the proteostasis.Failure or malfunction of the proteostasis network is often associated with human diseases.As a proteasome activator,REGγ plays an important role in many signaling pathways and the occurrence of diseases.Therefore,it has very important significance and potential application value of screening inhibitors of the interaction of REGγ and 20S proteasome.We found that the REGγ exerts its biological function through the combination of REGy and the 20S proteasome α7 subunit in preliminary mechanism research.We use the split-firefly luciferase fragment complementation technique to construct a set of monitoring system to quantify the interaction between REGy and α7 in vivo,that is,lucN(416-linkerl)-REGγ and α7-lucC(linkerl-394).But immunofluorescence results show that lucN-REGγ is mainly localized in the nucleus,while lucC-α7 is mainly localized in the cytoplasm.In order to further improve the screening effect of the system and to eliminate the interference of endogenous REGγ,We optimized the monitoring system:85 and 86 lysine(K)in the nuclear localization sequence of REGγ are mutated to be glutamine(Q),the mutant lucN-REG(K85QK86Q)is mainly localized in the cytoplasm,which is co-located with the lucC-a7,so that the luciferase activity is also greatly enhanced.REGγ(NL)(delete of active loop of REGγ)identified by the screening system has the ability to inhibit the binding of endogenous REGγ and a7,which also reflects the feasibility of the screening system.In addition,REGγ exerts its biological functions mainly in the form of a heptamer ring.When the luciferase N terminal and C terminal fragment was applied to the REGγ,it will be a screening system for screening specific inhibitors of REGy heptamer.These two systems have laid the solid foundation for the effective screening the inhibitors of REGy in the future.