Protein stability regulators screening assay (Pro-SRSA): protein degradation meets the CRISPR–Cas9 library
© The Author(s) 2016
Received: 6 June 2016
Accepted: 13 June 2016
Published: 29 June 2016
The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled “A genome-scale CRISPR–Cas9 screening method for protein stability reveals novel regulators of Cdc25A,” recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR–Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our findings, we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.
KeywordsCRISPR–Cas9 screening Protein stability Cdc25A Ubiquitination Acetylation
Fine-tuned degradation of proteins plays a crucial role in a variety of physiological conditions, including cell cycle progression, cell growth, differentiation, and signaling transduction. The inappropriate degradation of some cancer-related proteins may induce cellular transformation and tumorigenesis . Thus, understanding the precise regulation of protein degradation, especially for oncogenes and tumor suppressors, is important not only for understanding the biological mechanisms of cancer but also for developing therapeutic interventions. However, due to the lack of an efficient method, an unbiased and universal delineation of the regulators of protein homeostasis has not been previously possible. But now, based on the study reported in the article entitled “A genome-scale CRISPR–Cas9 screening method for protein stability reveals novel regulators of Cdc25A,” which was published in Cell Discovery, such an efficient method has been developed . In this study, our team devised a protein stability regulators screening assay (Pro-SRSA) to screen for regulators of protein stability by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR–Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing.
Using Pro-SRSA screening for cell division cycle 25A (Cdc25A) regulators, we found that cullin 4B (Cul4B)–damage-specific DNA binding protein 1 (DDB1)—DDB1 and CUL4 associated factor 8 (DCAF8) complex (Cul4B-DDB1DCAF8) is an E3 ligase for the degradation of Cdc25A. More interestingly, the screening also revealed that acetylation of Cdc25A at lysine 150, which is acetylated by E1A binding protein p300/CREB binding protein (p300/CBP) and deacetylated by histone deacetylase 3 (HDAC3), stabilizes the protein. In addition, using p53 as another example in our Pro-SRSA, we discovered a new degradation mechanism for p53 (manuscript in preparation).
Pro-SRSA is a powerful screening method that could help researchers better understand cellular protein regulation in a variety of physiological and/or pathological processes, especially for the abnormal expression of oncogenes and tumor suppressors during tumorigenesis. Genome-scale screening for regulators of protein stability may open many new avenues for the treatment, perhaps even the curing, of diseases. Thus, we are confident that this easy and efficient method would be helpful for researchers focusing on protein degradation.
YZW drafted the manuscript. TBK reviewed and revised the manuscript. Both authors read and approved the final manuscript.
We thank all members of the Kang’s Lab for critical discussions.
The authors declare that they have no competing interests.
Availability of data and materials
The GeCKO lenti-CRISPR library is available on Addgene (Feng Zhang’s Lab).
This work was supported by grants from the Key Project of Guangzhou (No. 1561000151), the Yangtze River Scholarship (No. 85000-52121100), the National Nature Science Foundation in China (No. 81530081, 31571395), and the 973 project (No. 2012CB967000).
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