了解遗传编码的标签如何影响异染色蛋白HP1α的相分离
bioRxiv : the preprint server for biology
|December 18, 2023
概括
用光蛋白标记异性染色蛋白HP1α会影响其液体-液体相分离 (LLPS) 特性. 像UnaG这样的较小标签最小地扰乱HP1αLLPS,使得体外-体内相关性更好,而较大的标签可以抑制或促进它.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 细胞生物学 细胞生物学
背景情况:
- 液-液相分离 (LLPS) 是细胞凝聚物的基础,由多价值相互作用驱动.
- 异染色蛋白蛋白1α (HP1α) 与染色素进行LLPS,并形成细胞点.
- 光标记在体内可视化HP1α动态时很常见,但其对内在相位分离的影响尚不清楚.
研究的目的:
- 系统地评估不同尺寸和链接长度的C端光标签如何影响HP1α与DNA相位分离.
- 为了评估HP1α相分离的体内研究中不同标签的实用性.
- 为了研究细胞拥挤对HP1α相分离的作用,使用聚乙烯糖醇 (PEG) 在体外.
主要方法:
- 使用各种C端标签进行HP1α与DNA的体外相位分离试验:AID-sfGFP (52kDa),mEGFP (27kDa) 和UnaG (13kDa) 具有16个氨基酸链接器.
- 通过变化蛋白质度来评估相分离能力.
- 添加拥挤剂 (10%的PEG8000或PEG4000),以模仿细胞拥挤,并观察HP1α和马尔托结合蛋白 (MBP) 阶段分离的影响.
主要成果:
- 大的AID-sfGFP标签促进了HP1α相分离,可能是由于无序的AID降解.
- mEGFP标签抑制了HP1α相分离,而UnaG标签显示了最小的扰动.
- 聚乙烯甘醇 (PEG) 的添加在拥挤条件下挽救了GFP标记HP1α的相分离,但也减少了野生型和突变HP1α之间的差异,并诱导了MBP的相分离.
结论:
- 标签选择对于在体外和体内研究HP1α相分离至关重要,UnaG为细胞和生化研究的相关性提供了一个不那么令人不安的选择.
- 像PEG这样的细胞拥挤剂可以诱导或增强宏分子的相分离,包括MBP这样的标签.
- 结果警告在存在基于PEG的拥挤剂时观察到的过度解释相位分离现象,因为它们可以改变潜在的相互作用动态.
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