一个全基因组的CRISPR/Cas9屏幕确定了calreticulin作为ATF6α的选择性抑制剂
Joanne Tung1, Lei Huang1, Ginto George1
1Cambridge Institute for Medical Research (CIMR), University of Cambridge, Cambridge Biomedical Campus, Cambridge, United Kingdom.
eLife
|July 29, 2024
概括
卡莱蒂库林 (CRT) 作为激活转录因子6α (ATF6α) 信号的内细胞网膜 (ER) 抑制剂. 这一发现揭示了展开蛋白质反应 (UPR) 的新调节机制.
科学领域:
- 细胞生物学 细胞生物学
- 分子生物学分子生物学
- 生物化学 生物化学
背景情况:
- 激活转录因子6 (ATF6) 是细胞内膜网膜 (ER) 应激的一个关键传感器,对未折叠蛋白质反应 (UPR) 至关重要.
- 涉及复杂的贩运途径的ATF6α信号的精确规范尚未完全理解.
- 了解ATF6α调节对于适应长期ER压力至关重要.
研究的目的:
- 使用全基因组CRISPR/Cas9屏幕系统地识别调节ATF6α信号的遗传因素.
- 阐明候选基因在ER压力条件下ATF6α激活中的特定作用.
- 揭示UPR途径的新型监管机制.
主要方法:
- 一个双UPR记者CHO-K1细胞系 (ATF6α/Inositol-requiring kinase 1 (IRE1)) 的生成.
- 无偏见的全基因组CRISPR/Cas9突变发生屏幕用于识别ATF6α的遗传调节者.
- 生物化学测定包括*体外*结合和共免疫沉以确认蛋白质相互作用.
- 分析ATF6α处理,贩运和记者基因活性在具有和没有卡尔雷蒂库林 (CRT) 的细胞中.
主要成果:
- 克里斯普尔屏幕识别了ATF6α激活的已知和新型调节者.
- 发现ER的伴侣卡尔雷蒂库林 (CRT) 能够选择性地抑制ATF6α信号传递.
- 缺乏CRT的细胞表现出构成ATF6α记者激活,增加BiP水平,并增强ATF6α的贩运/加工.
- CRT与ATF6α的光域直接相互作用,这表明它具有直接的调节作用.
- CRT 衰竭揭示了一个负反循环,ATF6α 通常基本上抑制 IRE1 活动.
结论:
- 卡尔雷蒂库林 (CRT) 不仅作为一个伴侣,而且作为ATF6α信号的特定抑制剂.
- 这确定了一种选择性调节UPRATF6α臂的新型机制.
- 这些发现揭示了UPR路径内的复杂交叉和反循环.
- 通过调节ATF6α活性,CRT在维持基底UPR平衡中发挥着至关重要的作用.
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