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Updated: Feb 10, 2026
Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Published on: March 21, 2022
A protein folding molecular imaging biosensor monitors the effects of drugs that restore mutant p53 structure and its
Ramasamy Paulmurugan1, Rayhaneh Afjei2, Thillai V Sekar1
1Cellular Pathway Imaging Laboratory (CPIL), Molecular Imaging Program at Stanford, Stanford University School of Medicine, Palo Alto, CA 94305, USA.
Abstract:
Misfolding mutations in the DNA-binding domain of p53 alter its conformation, affecting the efficiency with which it binds to chromatin to regulate target gene expression and cell cycle checkpoint functions in many cancers, including glioblastoma. Small molecule drugs that recover misfolded p53 structure and function may improve chemotherapy by activating p53-mediated senescence. We constructed and optimized a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) to determine small molecule-meditated folding changes in p53 protein. After initial evaluation of the biosensor in three different cells lines, we engineered endogenously p53P98L mutant (i.e. not affecting the DNA-binding domain) Ln229 glioblastoma cells, to express the biosensor containing one of four different p53 proteins: p53wt, p53Y220C, p53G245S and p53R282W. We evaluated the consequent phenotypic changes in these four variant cells as well as the parental cells after exposure to PhiKan083 and SCH529074, drugs previously reported to activate mutant p53 folding. Specifically, we measured induced RLUC complementation and consequent therapeutic response. Upon stable transduction with the p53 biosensors, we demonstrated that these originally p53P98L Ln229 cells had acquired p53 cellular phenotypes representative of each p53 protein expressed within the biosensor fusion protein. In these engineered variants we found a differential drug response when treated with doxorubicin and temozolomide, either independently or in combination with PhiKan083 or SCH529074. We thus developed a molecular imaging complementation biosensor that mimics endogenous p53 function for use in future applications to screen novel or repurposed drugs that counter the effects of misfolding mutations responsible for oncogenic structural changes in p53.
Insights
Researchers developed a novel biosensor to detect p53 protein folding changes, aiding in the discovery of new cancer drugs. This tool helps screen therapies targeting misfolded p53 mutations in cancers like glioblastoma.
Area of Science:
- Molecular Biology
- Cancer Research
- Drug Discovery
Background:
- Misfolding mutations in p53 impact cancer development, particularly glioblastoma, by altering gene regulation and cell cycle control.
- Small molecule drugs hold potential for cancer therapy by restoring p53 structure and function, thereby activating p53-mediated senescence.
- Developing tools to accurately assess p53 folding changes is crucial for identifying effective therapeutic strategies.
Purpose of the Study:
- To construct and optimize a split Renilla luciferase (RLUC) complementation molecular biosensor (NRLUC-p53-CRLUC) for detecting small molecule-induced p53 folding alterations.
- To engineer glioblastoma cells with endogenous p53 mutations to express this biosensor for evaluating p53 protein variants (p53wt, p53Y220C, p53G245S, p53R282W).
- To assess the phenotypic consequences and drug responses in engineered cells upon treatment with known p53-activating compounds and chemotherapeutics.
Main Methods:
- Construction and optimization of a split RLUC complementation biosensor for p53 protein folding.
- Engineering of p53 mutant Ln229 glioblastoma cells to stably express the p53 biosensor variants.
- Evaluation of drug responses (PhiKan083, SCH529074, doxorubicin, temozolomide) by measuring RLUC complementation and phenotypic changes.
Main Results:
- The engineered Ln229 cells successfully acquired cellular phenotypes representative of the expressed p53 variants.
- A differential drug response was observed in the engineered cells when treated with doxorubicin and temozolomide, alone or in combination with PhiKan083 or SCH529074.
- The biosensor effectively detected drug-induced folding changes in p53 proteins.
Conclusions:
- A functional molecular imaging complementation biosensor mimicking endogenous p53 activity was successfully developed.
- This biosensor enables the study of p53 protein folding and its functional consequences in a cellular context.
- The developed biosensor platform is suitable for screening novel or repurposed drugs aimed at correcting oncogenic p53 structural defects.
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