Identification of mutant p53-specific proteins interaction network using TurboID-based proximity labeling

Hu, Shuang, et al. “Identification of mutant p53-specific proteins interaction network using TurboID-based proximity labeling.” Biochemical and Biophysical Research Communications (2022). https://doi.org/10.1016/j.bbrc.2022.05.046

Abstract

Backgrounds

Although several studies on mutant p53 reported cancer-promoting activities via “gain-of-function”, the mechanism underlying these differences in function between p53 R175H, R175P, and p53 wild-type (WT) remains unclear.

Methods

Linking miniTurbo with p53 WT, R175H, and R175P, the expression of fusion and biotinylated proteins were assessed by Western blotting. The function and subcellular localization of fusion proteins were detected by apoptosis assay and immunofluorescence, respectively. Biotinylated proteins were analyzed by liquid chromatography-tandem mass spectrometry, followed by bioinformatics analysis. Small-scale pull-downs and Co-Immunoprecipitation were performed to validate the interaction between mutant or p53 WT and biotinylated proteins.

Results

The fusion protein’s cellular localization and function were consistent with those of previous studies on the corresponding p53. Comparative profiles of R175H versus WT showed that most of the interacting proteins belonged to the intracellular organelle lumen, and the pathways involved were metabolism and genetic information processing. Comparative profiles of R175P versus WT suggested that the majority of the interacting proteins belonged to the intracellular organelle lumen and the extracellular membrane-bounded organelle, and the pathways involved were metabolism and genetic information processing pathways. The comparison between R175H and R175P revealed that most interacting proteins belonged to the organelle lumen, and pathways involved were genetic information processing pathways. Finally, the mutation of p53 significantly altered the interaction with the target proteins were confirmed.

Conclusion

We verified the reliability of the miniTurbo system and obtained candidate targets of mutant p53, which provided new thoughts on the mechanism of mutant p53 gain-of-function and new potential targets for cancer therapy.