GST pull-down联合质谱筛选(肌)营养不良短小蛋白结合蛋白1在小鼠睾丸组织中的相互作用蛋白质  

Searching for Dysbindin-1 Interacting Proteins in Mouse Testis by GST Pull-down and Mass Spectrometry

作　　者：张盼盼[1] 闫慧[1] 袁业锋 冯雅琴[1] 

机构地区：[1]山西医科大学组织胚胎学教研室,太原030001 [2]中国科学院遗传与发育生物学研究所,北京100101

出　　处：《中国生物化学与分子生物学报》2017年第3期286-293,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.30900777);山西省青年科技研究基金(No.2010021036-1);山西医科大学博士启动基金(No.03200804)资助~~

摘　　要：(肌)营养不良短小蛋白结合蛋白1(dystrobrevin binding protein 1,dysbindin-1)是溶酶体相关细胞器生物发生复合体-1(biogenesis of lysosome-related organelles complex 1,BLOC-1)的1个亚基,在多种组织细胞中广泛表达;然而,其在睾丸组织中的作用至今尚不明确。为寻找(肌)营养不良短小蛋白结合蛋白1在睾丸组织中的相互作用蛋白质,以进一步研究(肌)营养不良短小蛋白结合蛋白1在睾丸中的作用,本研究首先在Rosetta(DE3)菌种中表达可溶性GST-dysbindin-1融合蛋白,经谷胱甘肽-琼脂糖珠亲和纯化后,与小鼠的睾丸组织蛋白质孵育进行GST pull-down实验,并通过液相色谱串联质谱(LC MS/MS)分析筛选(肌)营养不良短小蛋白结合蛋白1在睾丸组织中的相互作用蛋白质。利用Bio GPS数据库聚类在睾丸组织中高表达和特异性表达的互作蛋白质,运用DAVID6.8在线分析工具从细胞组分、分子功能、生物学过程和KEGG通路等方面对筛选出的互作蛋白质进行GO(gene ontology)富集分析。本实验共筛选出108个(肌)营养不良短小蛋白结合蛋白1在睾丸组织中的潜在互作蛋白质,其中98个为尚未报道的(肌)营养不良短小蛋白结合蛋白1相互作用蛋白质,7个为睾丸高表达蛋白质,5个为睾丸特异性表达的蛋白质。这些候选蛋白质主要分布在细胞质、细胞核、细胞膜、细胞外泌体等细胞组分中,通过与蛋白质、核酸等分子结合参与蛋白质翻译和转运、囊泡运输及凋亡等生物学过程以及氨基酸生物合成、溶酶体及蛋白酶体等生物学通路。我们推测,在睾丸组织中(肌)营养不良短小蛋白结合蛋白1可能通过与多种蛋白质相互作用参与精子的发生和受精等过程。Dystrobrevin binding protein 1（ dysbindin-1）,a subunit of biogenesis of lysosome-related organelles complex 1（ BLOC-1） is widely expressed in a variety of tissues and cells. However,its roles in the testis are uncertain. In order to understand the roles of dysbindin-l in testis,experiments were peformed to obtain the dysbindin-1 associated proteins. GST-dysbindin-1 fusion protein was expressed inthe Rosetta（ DE3） bacteria and purified by glutathione-Sepharose 4B. GST pull-down assay was performed by incubation of the purified GST-dysbindin-1 fusion protein with the testicular tissue lysate,followed by liquid chromatoqraphy-coupled tandem mass spectrometry（ LC MS / MS） to analyze and select the dysbindin-1 associated proteins. The highly and specifically expressed interacting proteins in the testis were analyzed and clustered by the Bio GPS 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,molecular function,biological processes and KEGG pathway. In this study,we enriched 108 potential dysbindin-1interaction proteins in the testis,98 of which were previously unreported dysbindin-1 interaction proteins,and 7 high testis expression proteins and 5 testis-specific proteins were found. These candidates mainly distributed in the cytoplasm,nucleus,cell membrane and extracellular exosome,played important roles in protein binding and RNA binding,which may be involved in multiple biological processes such as protein translation and transport,vesicle trafficking,apoptotic process,and biological pathways such as biosynthesis of amino acids,lysosome and proteasome. We speculate that dysbindin-1 may participate in the spermatogenesis and fertilization through interaction with multiple proteins in the testis.

关 键 词：(肌)营养不良短小蛋白结合蛋白1 睾丸组织 相互作用蛋白 功能分析 小鼠 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]