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Updated: Jun 11, 2026
Purification of Ubiquitinated p53 Proteins from Mammalian Cells
Published on: March 21, 2022
Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines
Mohd A Yusuf1, Trinette Chuang, G Jayarama Bhat
1Department of Biomedical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106, USA.
Abstract:
Previously, we reported that human p53 is functionally inactivated by S-glutathionylation at Cys-141 during oxidative and DNA-damaging treatments. Here, we describe the presence of thiolated p53 and the dynamic nature of this modification in human tissues using unique and specific polyclonal antibodies raised against a 12-residue p53 peptide bearing a mixed disulfide at Cys-141. The affinity- purified antibodies (glut-p53) were sequence-specific in that they recognized the antigenic peptide but not the unthiolated peptide or a scrambled glutathionylated peptide in ELISAs. On immunoblots, the purified antibodies did not react with native p53 or recombinant p53 (rp53), but readily detected the glutathionylated or cysteinylated or ethanethiol-treated rp53 only under nonreducing conditions. Untreated HCT116 cells showed low levels of glut-p53, which increased markedly after H(2)O(2), diamide, cisplatin, and doxorubicin treatments. Glut-p53 levels decreased sharply after cells were passed into oxidant-free medium, suggesting efficient dethiolation. The mutant p53 present in HT29 and T47D human cancer cells was also recognized. In vitro, the glut-p53 was rapidly degraded by rabbit reticulocyte lysates. Human prostate and prostate cancer tissues showed an abundant presence of glut-p53 in luminal epithelium, a site well known to generate ROS. Melanoma and colon cancer samples were also positive for glut-p53. The availability of the thiolation-specific antibodies should enhance our knowledge of p53 regulation in redox-perturbed states found in various diseases including cancer.
Insights
Researchers developed specific antibodies to detect thiolated p53 (glut-p53) in human tissues. This modification, linked to oxidative stress and cancer, is dynamic and present in various cancer types.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Human p53 protein is crucial in cellular response to DNA damage and oxidative stress.
- S-glutathionylation at Cys-141 was previously identified as a mechanism for p53 functional inactivation.
- Understanding the presence and dynamics of thiolated p53 in human tissues is vital for disease research.
Purpose of the Study:
- To develop and validate specific antibodies for detecting thiolated p53 (glut-p53) in human tissues.
- To investigate the presence and dynamic nature of p53 thiolation in various human cell lines and tissues.
- To explore the role of p53 thiolation in redox-perturbed states and disease, particularly cancer.
Main Methods:
- Generation of unique polyclonal antibodies against a p53 peptide modified with a mixed disulfide at Cys-141.
- Validation of antibody specificity using ELISAs and immunoblots with native, recombinant, and modified p53.
- Detection of glut-p53 levels in human cell lines (HCT116, HT29, T47D) under various stress conditions and in human tissue samples (prostate, melanoma, colon cancer).
Main Results:
- Newly developed affinity-purified antibodies (glut-p53) specifically recognize S-glutathionylated p53 under nonreducing conditions.
- Glut-p53 levels increase significantly in HCT116 cells upon exposure to oxidative and DNA-damaging agents (H2O2, diamide, cisplatin, doxorubicin) and decrease upon removal of the oxidant.
- Thiolated p53 is detected in various human cancer tissues, including prostate, melanoma, and colon cancer, as well as in mutant p53-expressing cancer cell lines.
Conclusions:
- The study successfully generated and validated antibodies for specific detection of thiolated p53.
- P53 thiolation is a dynamic modification present in human tissues, particularly in redox-active sites and cancer.
- These antibodies provide a valuable tool for advancing the understanding of p53 regulation in disease states involving oxidative stress.
