| Neurexin(Nrx)is a presynaptic transmembrane adhesion molecule.Its unique structure enables it to interact with multiple molecules before and after synapses to form transmembrane complexes and promote synaptic assembly and signaling.Neurexin and its ligands have been implicated in neurological disorders,including autism and schizophrenia.Therefore,the identification of Nrx ligands has guiding significance for understanding Nrx-related neurological diseases.Peroxidase-based proximity labeling(APEX2)is a technique that can label proteins in living cells and detect protein interactions.In this method,APEX2 and target protein can be fused and expressed by gene coding,which can directly capture transient or affinity interacting proteins.APEX2 has the advantages of fast catalytic efficiency and high sensitivity,and has been successfully applied to many model organisms such as mouse and Drosophila.In this study,APEX2 was fused with Nrx in Drosophila nervous system,and the molecular interaction with Nrx was screened through mass spectrometry and bioinformatics analysis.Firstly,we constructed transgenic flies HA-APEX2::Nrx,Nrx::HA-APEX2 and HA-APEX2,and verified the successful preparation of transgenic flies by three methods of genome,immunostaining and Western blot.APEX2 Nrx can be localized to the membrane of the neuromuscular junction(NMJ),and the number of synapse Boutons is not significantly different from Wild Type,suggesting that APEX2 does not interfere with Nrx function.HA-APEX2::Nrxand Nrx::HA-APEX2 respectively identified 145 and 222 proteins.HA-APEX2 in the control group detected 152 proteins.Fold Change≥2 or ≤1/2 was selected as the differentially proteins.GO enrichment analysis showed that the differential proteins of HA-APEX2::Nrx were mainly involved in biological processes such as organelle organization and cell development,and were associated with protein binding and presynaptic active regions.The proteins of Nrx::HA-APEX2 group are mainly membrane proteins,cell connection molecules and synaptic molecules,which are related to presynaptic active region,protein binding and signal transduction.KEGG signaling pathway analysis showed that APEX2 Nrx-labeled molecules were involved in metabolic pathways,genetic information processing,environmental signaling and other pathways.In this study,the APEX2,Affinity-purified mass spectrometry(AP-MS)and bioinformatics methods were used to screen the interaction molecules of Nrx in the nervous system of Drosophila.The Nrx spatio-temporal interaction proteome was successfully created,and the signal transduction pathways and biological events that Nrx might be involved in were analyzed,hoping to provide new targets and ideas for the treatment of Nrx-related neurological diseases. |
