白血病细胞中新型G_sα缺失体与CPP32的相互作用(英文)  

Interaction of a novel G_sα deletion mutant with CPP32 in leukemic cells

作　　者：叶棋浓[1] 姚潇[2] 王恒梁[1] 张述[3] 逯好英 苏国富[1] 黄翠芬[1] 周廷冲[4] 

机构地区：[1]北京生物工程研究所,北京100071 [2]陕西师范大学生物系,西安710062 [3]北京医科大学临床肿瘤学院,北京100036 [4]北京基础医学研究所,北京100850

出　　处：《中国实验血液学杂志》1998年第4期277-282,共6页Journal of Experimental Hematology

摘　　要：CPP32/Yama/Apopain/Caspase-3在不同形式的细胞凋亡途径中起核心作用。目前发现,CPP32可切割多种底物,但以CPP32为引诱蛋白用酵母双杂交系统分离与其相互作用的分子方面的研究,在国内外尚未见报道。为进一步了解肿瘤细胞信号转导途径,本文用该系统筛选与CPP32(作为引诱蛋白)相互作用的蛋白质。将CPP32引诱蛋白表达载体和肿瘤细胞文库质粒导入到酵母细胞HF7C中,用Trp-Leu-His-营养缺陷培养基初步筛选文库中插入cDNA所编码的序列与CPP32相互作用的菌落,再进一步检测β-半乳糖苷酶活性,结果从200多个Trp^+Leu^+His^+酵母菌落中获得7个LacZ^+阳性的酵母菌落。经分离阳性菌中的文库质粒和序列分析发现,在人白血病细胞株Jurkat中,新型G_sα缺失体(其正常蛋白与腺苷酸环化酶信号转导途径的激活相关)可与CPP32相互作用。纯化大肠杆菌中表达的G_sα缺失体及CPP32,用ELISA法进一步证实了这种相互作用。结论推测白血病细胞(至少某种白血病细胞)中CPP32参与了腺苷酸环化酶信号转导途径。CPP32/Yama/Apopain/Caspase-3 plays a central role in different apoptotic pathways. Although multiple protein substrates of CPP32 have been found, there are no reports on identification of proteins that interact with CPP32 (as a bait protein) using yeast two-hybrid system. In this study, the two-hybrid system, in which positive clones were isolated by His+ and Lac Z+ phenotypes, was used to screen for proteins interacting with CPP32, to further elucidate signal transduction pathway in tumor cells. The CPP32 bait protein expression vector and tumor cell library plas-mids were transformed into the yeast strain HF7C. The transformation mixture was plated on medium lacking Trp, Leu and His in the initial screen. Colonies growing on the selection medium were further assayed for β-galactosidase activity. From more than 200 Trp+ Leu+ His+ colonies, 7 LacZ+ colonies were obtained. DNA sequence analysis of the library plasmids from the positive colonies indicated that a novel deletion mutant of Gsα in Jurkat cells, whose normal type was reported to be involved in stimulation of adenylate cyclase pathway of signal transduction, interacted with CPP32 in the two-hybrid screen. Direct interaction of CPP32 with the deletion mutant was further confirmed in an ELISA assay with the purified proteins of CPP32 and the deletion mutant expressed in E. coli. Our results suggest that CPP32 may be involved in the adenylate cyclase transduction pathway in leukemic cells.

关 键 词：CPP32 G_sa缺失体 白血病细胞 细胞凋亡 

分 类 号：R733.7[医药卫生—肿瘤]