摘要
目的:探讨人蛋白酶体α亚基3型(PSMA3)和Bc1-2家族中的促细胞凋亡蛋白分子Bim之间是否存在相互作用,并初步了解PSMA3对细胞凋亡的影响。方法:借助PlexA-bimL技术从人胎脑PB42AD文库筛选PSMA3;借助显微共聚焦技术探讨PSMA3与BIM相互作用的亚细胞定位;应用免疫共沉淀技术、Western技术探讨PSMA3与BIM的相互作用;通过流式细胞仪技术初探PSMA3能否通过调节BIM来对细胞凋亡产生影响。结果:在人胎脑pB42AD文库用pLexA-bim L筛选得到121个阳性克隆,测序分析发现其中有11条基因重复1次以上。其中TUBB5、GRB10、MIF、FHC等基因都有文献报道其与细胞凋亡有直接关系;内共定位表明,PSMA3-RFP(红色)和GFP-BimL(绿色)的荧光分布区域主要存在于细胞胞浆区,并且在相同视野下的PSMA3-RFP和GFP-BimL的荧光分布图叠加,提示在细胞内PSMA3和Bim在空间上存在相互作用的可能性;免疫共沉淀结果表明,外源表达的PSMA3能与GFP-tagged BimL在HEK293细胞内相互作用,特别是在过表达Bim的凋亡细胞中。结论:PSMA3可以与Bim相结合并对细胞凋亡有一定的影响。
Objective : To explore the interaction between Proteasome Alpha Subunit 3(PSMA3) and pro-apoptotic proteins Bim of BC1-2family,and to understand effects of human Proteasome Alpha Subunit 3(PSMA3) on pro-apoptotic protein molecule Bim Methods : Screening out PSMA3from PB42AD by using the technology of plexA-bimL;finding out the subcellular localization as a result of interaction between PSMA3and BIM by using confocal microscope technology;researching the interaction between PSMA3and BIM through coimmunoprecipitation and Western technology;doing apreliminary study about whether Bim can be regulated by PSMA3to affect apoptosis by using the flow cytometer.Results : The result of screening : After screening PB42AD by plexA-bimL,we found 121positive clones.Sequence analysis verified that there were 11genes which were repeated more than once.And those genes included MIF,GRB10,TUBB5and FHC which have been reported to be directly involved in apoptosis.The results of co-localization of cells : The fluorescence area of PSMA3(red) and GFP-BimL(green) were mainly located in the cytoplasm.In the same horizon co-located areas(yellow) of the two protein molecules could be discovered.The fluorescence co-localization inferred the possible interaction between PSMA3and Bim in cells.The results of coimmunoprecipitation : In HEK293cells,PSMA3of exogenous expression could interact with GFP-tagged BimL,especially in those apoptotic cells of overexpression Bim.Conclusion : The PSMA3combined with Bim can have a certain impact on apoptosis.
