Study On The Mechanism Of Interaction Between Espin And Whirlin

Usher syndrome type II(USH2)causes blindness and deafness,and is the most common genetic disease affecting vision and hearing.Type Ⅱ(USH2)patients have congenital moderately severe hearing loss and normal vestibular reaction,and it is significant to study because USH2 is the most common type of Usher syndrome.There are three identified proteins underlying this Usher syndrome type Ⅱ: usherin,GPR98(G protein-couple receptor 98),and whirlin,They form the USH2 protein complex in vivo,which causes USH2 if any protein does not form the complex.Among them,usherin and GPR98 protein are mostly outside the cell membrane,and there is a PDZ binding domain(PBM)at the C terminal.whirlin is a scaffold protein containing 3 PDZ domains and a PR(proline-rich),whirlin acts as a link to the USH2 protein complex in the cell,which is necessary to form the USH2 protein complex.Proteins that interact with whirlin are thought to be candidate of the USH2 protein complex.It has been reported that both espin and whirlin are distributed in the microvilli of sensory cells and can interact with each other in vitro,but the mechanism of the interaction between whirlin and espin is not clear.In this paper,two whirlin fragments are constructed using restriction endonuclease and eukaryotic vectors,wherein the N-terminal fragment contains the first two PDZ domain and a PR,and the C-terminal fragment contains the last PDZ domain.In order to detect its corresponding protein products with espin protein in the cell positioning and interaction,we transfect the recombinant plasmid in COS-7 cells,then observe the localization of whirlin and espin in cells by different fluorescent.The recombinant plasmid was transfected into HEK293 T cells,co-immunoprecipitation was used to in the lysate of the cell.Finally,Western Blot was used to identify the interaction between whirlin and espin.The experiment found that the N-terminal fragment of whirlin had a co-location with espin and was able to pull down the espin in the cell lysis solution.We had divided whirlin into similar two in size,the N-terminal fragment containing the first two PDZ domain,and the C-terminal fragment contains PR and finally a PDZ domain structure,the two recombinant plasmid are not interact with espin.Compared with previous experimental data,the PR of whirlin and espin has important influence on their interaction.Although the PDZ domain is possible to directly participate in the interaction,PR seems to have an effect on the structure of the protein,thereby affecting the interaction of the proteins.Our findings provide a new insight on how USH2 complex is assembled and opens a new potential therapeutic path for USH2.