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

Area of Science:

• Molecular Biology
• Cancer Research
• Proteomics

Background:

• Mutant p53 exhibits gain-of-function activities in cancer, but the mechanisms differ between specific mutations (R175H, R175P) and wild-type (WT) p53.
• The precise functional distinctions and molecular underpinnings of these p53 variants remain largely unelucidated.

Purpose of the Study:

• To elucidate the distinct protein interaction profiles of p53 R175H and R175P mutants compared to p53 WT.
• To identify novel protein targets associated with mutant p53 gain-of-function.
• To explore potential therapeutic strategies for cancers driven by mutant p53.

Main Methods:

• Utilized the miniTurbo system to create fusion proteins with p53 WT, R175H, and R175P.
• Assessed fusion protein expression, localization, and function using Western blotting, apoptosis assays, and immunofluorescence.
• Identified interacting proteins via liquid chromatography-tandem mass spectrometry and bioinformatics analysis, validating interactions with pull-down and Co-Immunoprecipitation assays.

Main Results:

• Fusion protein behavior mirrored known p53 functions and localization.
• R175H and R175P mutations significantly altered interactions with proteins primarily located in the intracellular organelle lumen.
• Specific pathway analyses revealed differences in metabolism and genetic information processing between mutant and WT p53 interactions.

Conclusions:

• The miniTurbo system reliably identified candidate targets for mutant p53.
• Findings provide novel insights into the gain-of-function mechanisms of mutant p53.
• Identified potential new targets for developing cancer therapies.