【摘要】 筛选间隙连接蛋白31 (connexin31,Cx31) 相互作用蛋白并研究其在Cx31运输中的功能. 运用制备的抗Cx31多克隆抗体免疫沉淀,SDS-聚丙烯酰胺凝胶电泳分离,蛋白质条带回收,蛋白质胶块酶解,电喷雾-四极杆-飞行时间质谱分析,数据库扫描筛选可能相互作用蛋白,可能互作蛋白经免疫共沉淀、细胞免疫共定位等证实,确定actin为Cx31相互作用蛋白. 用药物处理细胞,抑制actin的功能,观察Cx31定位与间隙连接通道的通透性,确定actin在Cx31运输中的功能. 当药物抑制actin的功能时,Cx31很少能到达细胞膜上形成间隙连接通道,Cx31主要分布在胞质中;当药物抑制tublin的功能时,Cx31能到达细胞膜上形成间隙连接通道,细胞免疫荧光实验显示间隙连接斑有增多的现象,但染料转移实验表明细胞膜上间隙连接通道并没有增加. Actin在Cx31运输至细胞膜上形成间隙连接通道的过程中具有重要作用.更多还原

【Abstract】 Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Connexin31 (Cx31) is an important member of connexin β family. Mutations in Cx31 are associated with erythrokeratodermia variabilis(EKV), hearing impairment and peripheral neuropathy. The pathological mechanism for Cx31 mutants in these diseases remains unknown. Assembly, intracellular transport, plaque assembly and stability and channel conductivity of Cx31 are finely regulated and likely involve proteins that interact with Cx31. However, little is known about the Cx31 interaction proteins. A proteomics approach was applied to screen Cx31 binding proteins using HT1080 cells stably expressing a myc-tagged Cx31. Immunoprecipitation followed by peptide sequence analysis identified association of actin and Cx31. Interaction between actin and Cx31 is further confirmed by coimmunoprecipitation and immuno-colocalization. Pharmacological disruption of actin polymerization inhibits the plasma membrane localization of Cx31. The results suggested the actin cytoskeleton may play an essential role in regulating Cx31 trafficking via direct association.更多还原