Related Experiment Video
Updated: Apr 12, 2026
Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material
Published on: August 1, 2018
UBE2Q1 in a Human Breast Carcinoma Cell Line: Overexpression and Interaction with p53
Sayed Mohammad Shafiee1, Mozhgan Rasti, Atefeh Seghatoleslam
1Departments of Biochemistry- Recombinant Protein Laboratory, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract:
The p53 tumor suppressor protein is a principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage. p53 is a substrate for the ubiquitin-proteasome system, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination have not been well studied. UBE2Q1 is a novel E2 ubiquitin conjugating enzyme gene. Here, we investigated the effect of UBE2Q1 overexpression on the level of p53 in the MDA-MB-468 breast cancer cell line as well as the interaction between UBE2Q1 and p53. By using a lipofection method, the p53 mutated breast cancer cell line, MDA-MB-468, was transfected with the vector pCMV6-AN-GFP, containing UBE2Q1 ORF. Western blot analysis was employed to verify the overexpression of UBE2Q1 in MDA-MB-468 cells and to evaluate the expression level of p53 before and after cell transfection. Immunoprecipitation and GST pull-down protocols were used to investigate the binding of UBE2Q1 to p53. We established MDA-MB-468 cells that transiently expressed a GFP fusion proteins containing UBE2Q1 (GFP-UBE2Q1). Western blot analysis revealed that levels of p53 were markedly lower in UBE2Q1 transfected MDA-MB-468 cells as compared with control MDA-MB-468 cells. Both in vivo and in vitro data showed that UBE2Q1 co-precipitated with p53 protein. Our data for the first time showed that overexpression of UBE2Q1can lead to the repression of p53 in MDA-MB-468 cells. This repression of p53 may be due to its UBE2Q1 mediated ubiquitination and subsequent proteasome degradation, a process that may involve direct interaction of UBE2Q1with p53.
Insights
Overexpression of UBE2Q1, a novel ubiquitin-conjugating enzyme, reduces levels of the p53 tumor suppressor protein in breast cancer cells. This suggests UBE2Q1 may mediate p53 degradation through ubiquitination, impacting cancer cell growth.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- The p53 tumor suppressor protein is crucial for cellular responses to damage, regulating growth arrest, senescence, and apoptosis.
- p53 is targeted by the ubiquitin-proteasome system, but the specific ubiquitin-conjugating enzymes (E2s) involved in its ubiquitination remain largely uncharacterized.
- UBE2Q1 is identified as a novel E2 ubiquitin-conjugating enzyme.
Purpose of the Study:
- To investigate the impact of UBE2Q1 overexpression on p53 protein levels in the MDA-MB-468 breast cancer cell line.
- To explore the potential interaction between UBE2Q1 and p53.
- To elucidate the role of UBE2Q1 in the regulation of p53 stability.
Main Methods:
- Transfection of MDA-MB-468 cells with a vector expressing UBE2Q1 (GFP-UBE2Q1) using lipofection.
- Western blot analysis to confirm UBE2Q1 overexpression and assess p53 expression levels.
- Immunoprecipitation and GST pull-down assays to determine the interaction between UBE2Q1 and p53.
Main Results:
- Overexpression of UBE2Q1 in MDA-MB-468 cells led to a significant decrease in p53 protein levels compared to control cells.
- Both in vivo and in vitro experiments demonstrated co-precipitation of UBE2Q1 with p53 protein, indicating a direct interaction.
- The findings establish a novel role for UBE2Q1 in the regulation of p53.
Conclusions:
- UBE2Q1 overexpression represses p53 levels in MDA-MB-468 breast cancer cells.
- This repression is likely mediated by UBE2Q1-dependent ubiquitination of p53, leading to its subsequent proteasome degradation.
- The direct interaction between UBE2Q1 and p53 suggests a mechanism for targeted protein degradation in cancer cells.
