通过基于遗传密码扩展技术的FRET系统分析单个活细胞中的蛋白质相互作用
Seong-Hyun Park1, Wooseok Ko2, Hyun Soo Lee2
1Center for Biofunctional Molecules, Department of Chemistry , Yonsei University , Seoul 03722 , Republic of Korea.
Journal of the American Chemical Society
|February 2, 2019
概括
热冲击蛋白70 (Hsp70) 结合巴克斯抑制细胞灭绝. 新的FRET方法揭示了诱导细胞灭绝的物质如何破坏这种Hsp70-Bax相互作用,为细胞死亡调节提供了洞察力.
科学领域:
- 分子生物学
- 细胞生物学
- 生物化学
背景情况:
- 热冲击蛋白70 (Hsp70) 是细胞灭亡的关键调节剂,已知它能结合巴克斯并抑制细胞的编程死亡.
- 在诱导亡过程中,Hsp70-Bax复合物解离的确切机制尚未完全理解.
研究的目的:
- 阐明巴克斯在亡过程中与Hsp70脱离的分子机制.
- 开发和使用一种新的Förster共振能量传输 (FRET) 系统,用于实时研究活细胞中的蛋白质相互作用.
主要方法:
- 使用与黄色光蛋白 (YFP) 融合的Hsp70和通过遗传密码扩展与光氨基酸 (ANAP) 工程的BAX的FRET系统的开发.
- 活细胞成像和FRET信号的时间依赖性分析以监测Hsp70-Bax相互作用.
- 研究各种诱导细胞灭绝的物质,包括巴克斯激活剂,p53激活剂,死亡配体和Bcl-2抑制剂对Hsp70-Bax复合物的作用.
主要成果:
- 针对BAX触发部位的BAX激活剂抑制了Hsp70-BAX相互作用,而针对C端S184部位的特定激活剂则没有.
- 抑制Hsp70- Hsp40相互作用也阻断了Bax- Hsp70相互作用.
- 通过涉及p53的活性化或仅BH3蛋白的活性化,促进了巴克斯与Hsp70的解离.
结论:
- 这项研究成功地使用了一种新型FRET系统来剖析Hsp70-Bax复合物解离过程中的机制.
- 不同的亡诱导剂利用不同的分子途径来破坏Hsp70-Bax相互作用,突出显示了亡调节的复杂性.
- 开发的FRET系统是研究生物系统中的其他动态蛋白质相互作用的宝贵工具.
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