In a recent study published in the Cell Reports journal, researchers assessed the impact of protein adduction on p53 tumor suppressor inhibition.
Studies have shown that exposing esophageal cells to components of reflux increases reactive oxygen species (ROS) levels, resulting in the generation of isolevuglandins (isoLG), which are products of lipid peroxidation, forming several protein adducts within the esophageal epithelial cells of gastroesophageal reflux disease (GERD) patients. The specific isoLG-affected proteins formed due to reflux are yet unknown.
The preliminary screening analysis performed by the researchers indicated that adducted proteins include a component called the p53 tumor suppressor. However, it is unclear how reflux-generated isoLGs impact adducted proteins like p53.
About the study
In the present study, researchers explored the regulation of p53 in precancerous lesions before mutations within the p53 gene.
Since studies have shown the importance of the p53 protein in deoxyribonucleic acid (DNA) damage response (DDR), regulation, and DNA damage activation, the team assessed control and ABS-treated cells for p53 protein expression. To determine if reflux factors influenced other proteins generated due to DNA damage, the team examined the p73 protein. The p73 protein was assessed since it has several structural as well as functional similarities to p53. Furthermore, the PG13-Luc p53 luciferase reporter, which comprised several p53 binding sites repeats in its promoter, was assessed. As a control, the mutant p53 reporter MG15-Luc was employed.
The team then assessed whether isoLGs interacted with the p53 protein when exposed to reflux conditions. This was achieved by immunoprecipitation of the p53 protein from epithelioma papulosum cyprini (EPC)-2 and Barrett's esophagus cell line (CP-A) cells treated with either only acidic bile salts (ABS) or along with 2-hydroxy benzylamine (2-HOBA). The immunoprecipitated p53 protein was assessed for isoLG adducts via western blotting. p53 protein acetylation was examined in the EPC-2 and EC-A cells via western blotting employing the specific antibody recognizing acetylated p53.
Because of the hydrophobic features of isoLGs and their protein reactivity, they may impact the functional and structural characteristics of adducted proteins. Hence, the team determined if isoLGs impacted the structural features of p53. This was achieved by using the p53 antibody PAb 240, which identified the evolutionarily conserved epitope associated with the p53 protein, as a marker of misfolded p53 protein.
The study results showed that even when DNA damage was present, ABS did not considerably affect p53. On the other hand, camptothecin robustly caused p53 protein upregulation, indicating that ABS could influence p53 and DDR. As opposed to p53, ABS-elicited DNA damage resulted in robust p73 upregulation in the same cells. This confirmed that ABS had a selective inhibitory effect on p53. Furthermore, the team found that p73 inhibition resulted in a remarkable decline in messenger ribonucleic acid (mRNA) expression associated with these target genes, indicating that p73 impacted their upregulation. Also, ABS significantly affected the p53 pathway, thus inhibiting the expression of several genes.
The team found that 2-HOBA treatment recovered the transcription profile associated with the p53 signaling pathway. This suggested that isoLGs can influence p53 regulation. Notably, no effects of 2-HOBA were detected on pH or other parameters. Analysis of endogenous p53 protein's interaction with the dual-luciferase reporter assay, the team revealed that ABS considerably inhibited p53 activity and 2-HOBA alleviated inhibition induced by ABS.
The next-generation sequencing (NGS) plots demonstrated that 2-HOBA restored the p53 binding in ABS-treated CP-A cells, such as that of the PUMA(BBC3) and p21(CDKN1A) genes. Altogether, the team noted that isoLGs modulated the binding of the DNA to the p53 protein along with its transcription activity. Furthermore, ABS treatment remarkably increased p53-isoLG protein adducts concentrations, while 2-HOBA considerably inhibited their formation. Similarly, p53 adduction along with isoLG was also verified via CP-A cells treated with chemically synthesized isoLGs.
The team found that even in the presence of ABS-induced DNA damage, p53 acetylation did not show a significant increase in comparison to control untreated cells. This was in contrast to camptothecin (CAMP) which robustly increased p53 protein acetylation. Furthermore, 2-HOBA reversed the inhibitory impacts of ABS while elevating p53 acetylation in the presence of ABS-induced DNA damage. This suggested that isoLG adduction could influence p53 protein acetylation.
2-HOBA could also inhibit conformational alterations within the p53 molecule present in the EPC2 and CP-A, which suggested that ABS elicited conformational alteration, which could lead to the exposure of the intramolecular PAb 240 epitope of p53. Additionally, the p53 protein could form high molecular weight aggregates, which were detectable by the D01 or PAb 240 antibodies. The p53 protein aggregate having high molecular weight was also observed in the isoLGs- and ABS-treated CP-A cells when assessed using the p53 D01 antibody and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). However, the team found no protein aggregates in cells exposed to ABS along with 2-HOBA.
Overall, the study findings demonstrated the aggregation and inactivation of the p53 protein via isoLG adduction that could eventually result in carcinogenic changes. This mechanism could be inhibited by the isoLG scavenger, 2-HOBA, which effectively prevented the generation of isoLG protein adducts as well as restored the p53 protein.
- Ravindran Caspa Gokulan, Kodisundaram Paulrasu, Jamal Azfar, Monica Garcia Buitrago, Sergey I. Dikalov, Alexander I. Zaika. (2023). Protein adduction causes non-mutational inhibition of p53 tumor suppressor. Cell Reports. doi: https://doi.org/10.1016/j.celrep.2023.112024 https://www.cell.com/cell-reports/fulltext/S2211-1247(23)00035-9
Posted in: Medical Science News | Medical Research News | Disease/Infection News
Tags: Acetylation, Antibodies, Antibody, Assay, Barrett's Esophagus, Bile, cAMP, Cell, Cell Line, DNA, DNA Damage, Electrophoresis, Gastroesophageal Reflux Disease, Gel Electrophoresis, Gene, Genes, Immunoprecipitation, Luciferase, Molecule, Oxygen, p53 Protein, pH, Promoter, Protein, Protein Expression, Ribonucleic Acid, Signaling Pathway, Transcription, Tumor
Bhavana Kunkalikar is a medical writer based in Goa, India. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Her educational background allowed her to foster an interest in anatomical and physiological sciences. Her college project work based on ‘The manifestations and causes of sickle cell anemia’ formed the stepping stone to a life-long fascination with human pathophysiology.
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