| 李靓云唐超陈霞李建民△.RPAP3 蛋白中TPR 结构域的功能研究[J].,2012,12(12):2245-2249 |
| RPAP3 蛋白中TPR 结构域的功能研究 |
| Functional Study of TPR Domain in RPAP3 |
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| DOI： |
| 中文关键词: TPR 结构域 亚细胞定位 蛋白相互作用 细胞周期 |
| 英文关键词: TPR domain Subcellular localization Protein interaction Cell cycle |
| 基金项目:国家自然科学基金资助项目（30971092） |
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| 中文摘要: |
| 目的：构建针对RPAP3 TPR 区域的慢病毒载体，观察过表达对细胞周期的影响。方法：通过生物信息学软件比较RPAP3 结
构域组成，推测功能；分析RPAP3 核定位信号，构建瞬时表达质粒pEGFP-N2-RPAP3，激光共聚焦显微镜观察RPAP3 蛋白的亚
细胞定位；通过酵母双杂交和GST-Pulldown 实验研究RPAP3 与HSP70 的相互作用及作用靶点；构建慢病毒载体pLJM.
1-RPAP3，转染293T 细胞，收病毒感染MCF7 细胞，嘌呤霉素筛选获得稳定转染细胞系，流式细胞分析对细胞周期的影响。结果：
RPAP3 在多物种广泛存在，有高度保守性；蛋白存在典型核定位信号，激光共聚焦显微镜下，GFP 标记的RPAP3 蛋白主要分布在
细胞核；酵母双杂交和GST-Pulldown 实验证实RPAP3 与HSP70 间存在相互作用，且作用发生在RPAP3 的三联TPR 结构域和
HSP70 的GPTIEEVD 末端之间；流式细胞显示RPAP3 TPR 区域的高表达阻滞细胞周期且凋亡增加。结论：RPAP3 与HSP70 间
的相互作用发生在RPAP3 的三联TPR 结构域和热休克蛋白70 的GPTIEEVD 末端之间；构建高表达细胞株发现其对细胞周期
及凋亡有影响。 |
| 英文摘要: |
| Objective: To construct the RPAP3 (TPR domain)-containing lentivirus, and investigate the influence of the overexpression
of PRAP3 on cell cycle. Methods: RPAP3 with other homology proteins were compared by using sequence analysis tools. Then
RPAP3's nuclear localization signal sequence was analyzed, and produced transient expressing plasmid and its subcellular localization
was studied by laser scanning confocal microscope. The Yeast two-hybrid and GST-Pulldown we used To invest the coaction of RPAP3
with HSP70 and the reaction site,.localization by laser scanning confocal microscope. To invest the coaction of RPAP3 with HSP70 and
the reaction site, we used the Yeast two-hybrid and GST-Pulldown. A lentivirus expression vector wAS constructed, and infected MCF7
cells. Flow cytometry was used to to investigate the effect on cell cycle. Results: RPAP3 exists among many species and was highly
conserved. It has a typical nuclear localization single (RKKKKGKAKESSKKTRE) and is mainly distributed in the cell nucleus under laser
scanning confocal microscope. The interaction was confirmed , which happens between the three-TPR domain and hsp70 terminal
GPTIEEVD by Yeast two-hybrid and GST-Pulldown. The results of flow cytometry analysis showed that the overexpression of RPAP3
inhibit the cell cycle and increased apoptosis. Conclusions: RPAP3 can interact with hsp70 and hsp90 and it happens between the
three-TPR domain and hsp70 terminal GPTIEEVD. Overexpression of RPAP3 influenced the cell cycle and apoptosis. |
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