A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.
A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25A.
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用于蛋白质稳定性的基因组规模 CRISPR-Cas9 筛选方法揭示了 Cdc25A 的新型调节因子
DOI：
10.1038/celldisc.2016.14
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发表时间：
2016
期刊：
影响因子：
33.5
通讯作者：
Kang T
中科院分区：
文献类型：
--
作者：
Wu Y;Zhou L;Wang X;Lu J;Zhang R;Liang X;Wang L;Deng W;Zeng YX;Huang H;Kang T
稳定性的调节对于细胞中不稳定的蛋白质尤其重要。然而，一个方便和公正的方法来确定蛋白质稳定性的调节剂仍有待开发。最近，已经建立了一个基因组规模的CRISPR-Cas9文库，作为介导功能丧失筛选的遗传工具。在这里，我们通过将全基因组CRISPR-Cas9文库与基于双荧光的蛋白质稳定性报告基因和高通量测序相结合，开发了一种蛋白质稳定性调节剂筛选测定（Pro-SRSA），以筛选蛋白质稳定性的调节剂。以Cdc 25 A为例，Cul 4 B-DDB 1DCAF 8被鉴定为Cdc 25 A的新的E3连接酶。此外，Cdc 25 A在赖氨酸150处的乙酰化，其被p300/CBP乙酰化并被HDAC 3去乙酰化，阻止了蛋白酶体对Cdc 25 A的泛素介导的降解。这是第一项报道乙酰化作为一种新型的翻译后修饰，调节Cdc 25 A稳定性的研究，我们认为这种在基因组规模上无偏倚的CRISPR-Cas9筛选方法可以广泛用于全球范围内识别蛋白质稳定性的调节因子。
The regulation of stability is particularly crucial for unstable proteins in cells. However, a convenient and unbiased method of identifying regulators of protein stability remains to be developed. Recently, a genome-scale CRISPR-Cas9 library has been established as a genetic tool to mediate loss-of-function screening. Here, we developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome CRISPR-Cas9 library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Using Cdc25A as an example, Cul4B-DDB1DCAF8 was identified as a new E3 ligase for Cdc25A. Moreover, the acetylation of Cdc25A at lysine 150, which was acetylated by p300/CBP and deacetylated by HDAC3, prevented the ubiquitin-mediated degradation of Cdc25A by the proteasome. This is the first study to report that acetylation, as a novel posttranslational modification, modulates Cdc25A stability, and we suggest that this unbiased CRISPR-Cas9 screening method at the genome scale may be widely used to globally identify regulators of protein stability.