(307c) Construction of Neurexin Biosensor and Screening of Potential Ligands

Neurexins are neuronal presynaptic proteins that bind to the postsynaptic protein neuroligin at the synaptic junction. Both neurexins and neuroligins are involved in transmitting signals across the neural synapse. Many neurodevelopmental disorders involve abnormal synaptic function. The Neurexin-neuroligin interaction appears to play a key role in autism and other mental disorders. We have constructed a protein-based biosensor that can detect chemical ligands that bind to a given receptor protein. Our engineered bacterial biosensor fusion protein is expressed from a bacterial plasmid DNA, and consists of multiple protein domains including a solubility/stability platform (Maltose Binding protein and an engineered intein domain) and a reporter gene (phage T4 thymidylate synthase - TS) fused to a ligand-binding domain (LBD). The TS assay involves expression of this fusion protein in thymine-knocked out E. coli cells (D1210 ΔthyA) in a thymine-less media. Binding of the ligand in turn activates TS which is required for DNA synthesis in this E. coli strain. By measuring the growth phenotype of these cells at different ligand concentrations, we can calculate the EC50 for each of the ligands binding to Neurexin 2b. The biosensor system successfully developed in our lab has been previously used with estrogen receptors, thyroid receptors and Peroxisome Proliferator-Activated Receptor γ (PPARG), and has been published elsewhere.  Here we describe the construction of a biosensor variant in which Neurexin 2b serves as an LBD within the intein domain, such that binding of ligands is transduced into a growth phenotype via TS.  Using this neurexin-LBD biosensor strain, we found that Rosiglitazone, an anti-diabetic drug belonging to the thiazolidinedione class of drugs, causes a growth phenotype at low ligand concentrations, suggesting strong binding. This general approach could help identify ligands of Neurexin that interfere with the signaling across the synapse. Further, this might lead to a better understanding of the mechanism behind autism and other neural disorders.