Ppp5c 和TTC16 基因的TPR 结构域与Hsp70、Hsp90 蛋白 的相互作用及对细胞周期的影响

目的:研究Ppp5c及TTC16基因的TPR(tetratricopeptide repeat)结构域短片段和Hsp70及Hsp90家族蛋白的相互做用,及其过表达对细胞周期的影响。方法:通过生物信息学的分析及PCR的方法,克隆Ppp5c及TTC16基因的TPR结构域以及HSPA1A、HSP90AA1的全长基因,并连入酵母双杂交载体,通过ClonTech的酵母双杂交实验体系研究蛋白和蛋白之间的相互作用。把Ppp5c及TTC16基因的TPR结构域克隆入真核表达载体,构架稳定表达Ppp5c及TTC16基因的TPR结构域的MCF-7细胞系,并通过流式细胞实验观察细胞周期。结果:Ppp5c及TTC16基因的TPR结构域能与HSPA1A或HSP90AA1发生相互作用。Ppp5c及TTC16基因的TPR结构域在MCF-7中的过表达能严重影响细胞周期,引起细胞凋亡和S期阻滞。结论:本实验初步揭示了不同蛋白的TPR结构域在与Hsp70及Hsp90蛋白的相互作用性质的异同点以及其过表达对细胞周期的影响,为全面理解TPR结构域的功能、Ppp5c以及TTC16蛋白在细胞内的功能奠定了前期实验基础。

The Interactions of TPR Domains of Ppp5c/TTC16 with Hsp70/90 and Their Influence on the Cell Cycle

Objective: To investigate the interactions between the TPR(tetratricopeptide repeat) domains of Ppp5c/TTC16 and protein of Hsp70/Hsp90,to study the effect of the overexpression of TPR domains of Ppp5c/TTC16 on cell cycle in MCF-7 cell lines.Methods: After being analyzed by online informatics tools,the TPR domains of Ppp5c/TTC16 and full-length of HSPA1A/HSP90AA1 genes were respectively sub-cloned into two separate vectors of the yeast two-hybrid system provided by ClonTech to confirm the protein-protein interactions.The TPR domains of Ppp5c/TTC16 were sub-cloned into eukaryotic expression vectors which were stably transfected into MCF-7 cell-lines,whose cell cycle was then examined by flow cytometry.Results: The TPR domains of Ppp5c/TTC16 interacted with proteins of HSPA1A/HSP90AA1.A large amount of apoptosis was detected when the TPR domains of Ppp5c over-expressed in MCF-7 cell line,and the cell cycle was blocked at the S phase when the TPR domains of TTC16 over-expressed in MCF-7 cell line.Conclusion: This research revealed the discrepancy in the capacity of the combination of dissimilar TPR domains with Hsp70/Hsp90 proteins,as well as the effect of overexpression of TPR domains on the cell-cycle of MCF-7 cell line,which may help draw the panoramic picture of the functions of TPR domains and deep the understanding of intra-cellular functions of Ppp5c and TTC16 proteins.