Identifying And Searching For Dysbindin-1 Interacting Proteins In Mouse Testis

Objective:The aim of this study is to investigate and identify the interacting proteins of dysbindin-1 in order to explore the role of dysbindin-1 in testis.Methods:GST-dysbindin-1 fusion protein was induced in the Rosetta(DE3)strain,was purified by Glutathione-Sepharose-4B,GST pull-down assay was performed by incubating the purified GST-dysbindin-1 with testicular tissue lysates,and analyzed by liquid chromatography-coupled tandem mass spectrometry(LC MS/MS).The highly and specifically expressed interacting proteins in the testis were clustered by the BioGPS database.Using DAVID(The Database for Annotation,Visualization and Intergrated Discovery)6.8 online analysis tools,GO(Gene Ontology)functional analysis of dysbindin-1 interacting proteins was categorized based on cellular component(CC),molecular function(MF),biological process(BP)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway.Selecting the potential and interesting interaction protein vps4 a,the prokaryotic and eukaryotic expression vectors were constructed by molecular cloning technique,and the interaction between dysbindin-1 and vps4 a was verified by GST pull-down and immunoprecipitation.Results:1.In this study,we enriched 108 potential dysbindin-1 interaction proteins in the testis,98 of which were previously unreported dysbindin-1 interaction proteins,and 7 testis high expression proteins and 5 testis-specific proteins were found.2.DAVID 6.8 online analysis tools GO enrichment analysis found that these candidates were mainly distributed in the cytoplasm,nucleus,cell membrane and extracellular exosome,played role in protein binding and RNA binding,which may be involved in multiple biological process such as protein translation and transport,vesicletrafficking and apoptotic process,and several biological pathways such as biosynthesis of amino acids,lysosome,proteasome.3.Dysbindin-1 interacted with vps4 a was identified by GST pull-down and immunoprecipitation.Conclusion:Dysbindin-1 may participate in the spermatogenesis and fertilization through interaction with multiple proteins in the testis.