非晶状体βγ-晶状体蛋白与三叶因子蛋白复合物的细胞核转运及其选择性杀伤肿瘤细胞机制的研究  

Mechanism of βγ-CAT Cell Nuclear Transportation and Selectively Killing of Tumor cells

作　　者：何英英[1] 刘树柏[1] 钱金桥[1,2,3] 李文辉[1] 张云[1] 

机构地区：[1]中国科学院昆明动物研究所动物模型与人类疾病机理重点实验室 [2]中国科学院研究生院,北京100049 [3]昆明医学院第一附属医院麻醉科,云南昆明650032

出　　处：《Zoological Research》2008年第4期386-398,共13页动物学研究（英文）

基　　金：国家自然基金(30630014;30570359);中国科学院重要研究方向基金(KSCX2-YW-R-088)

摘　　要：非晶状体βγ晶状体蛋白与三叶因子蛋白复合物(βγ-CAT)是从大蹼铃蟾(Bombina maxima)皮肤分泌物中分离的分子量为72kDa的天然蛋白复合物。本研究通过激光共聚焦显微镜和Western blot分析βγ-CAT在人脐静脉内皮细胞(HUVEC)中的细胞核转运机制,以及βγ-CAT对多株肿瘤细胞(HCT116,HT29,A375,Hela,THP-1等)的细胞毒效应。结果表明:βγ-CAT的α亚基中含有典型的GTP/ATPase的保守结构模体Walker A和Walker B,体外检测到βγ-CAT具有GTP/ATP水解酶和GTP/ATP结合活性。在细胞核转运过程中,βγ-CAT的α亚基和β亚基参与形成约150kDa含有泛素化修饰信号的大分子复合物,且泛素化修饰信号和βγ-CAT的α亚基和β亚基共定位于细胞内和融合于细胞核区域的转运囊泡小体中。βγ-CAT能够选择性的杀伤肿瘤细胞,诱导肿瘤细胞脱落和发生凋亡。上述结果为进一步深入研究βγ-CAT的细胞核转运和调节细胞功能的分子作用机制提供思路和线索。βγ-CAT is a naturally existing protein complex of non-lens βγ-crystallin and trefoil factor, purified from Bombina maxima skin secretions. In HUVECs, βγ-CAT can be rapidly endocytosed via intracellular vacuole formation and translocated to the nucleus to regulate cell ruction. In this paper, we found that it contains conserved Walker B motifs （IILYDEPS, residues 6-13） and Walker A motifs （GQSLSGKS, residues 96-103） in the βγ-CAT α-subunit sequence. βγ-CAT showed potential NTP-binding and weak GTPase/ATPase activities in vitro. Through Western blotting analysis, we found that the α- and β-subunits of βγ-CAT participated in a 150kDa SDS-stable protein complex formation, which also contained positive ubiquitination signals in the βγ-CAT treated HUVEC. Furthermore, under confocal microscopy, the immunofluorescence signals of ubiquitin and βγ-CAT subunits were co-localized in the vacuoles that were distributed in the cytoplasm and nucleus. In addition, βγ-CAT could induce several tumor cell＇s detachment and apoptosis, and selectively kill tumor cells. These findings provide a clue to understand the mechanism of βγ-CAT endocysis and nuclear transport, and give an insight to investigate the possible occurrence of similar molecule＇s cellular functions and action mechanisms of non-lens βγ-crystallins and trefoil factors in mammals.

关 键 词：非晶状体βγ-晶状体蛋白 三叶因子 非晶状体βγ-晶状体蛋白和三叶因子蛋白复合物 GTP/ATP水解酶 肿瘤细胞脱落与凋亡 泛素化修饰 细胞核定位 

分 类 号：R73-3[医药卫生—肿瘤]