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Studying Homo-oligomerization and Hetero-oligomerization of MDMX and MDM2 Proteins in Single Living Cells by Using In Situ Fluorescence Correlation Spectroscopy
- Source :
- Biochemistry. 60:1498-1505
- Publication Year :
- 2021
- Publisher :
- American Chemical Society (ACS), 2021.
-
Abstract
- Protein oligomerization plays a very important role in many physiological processes. p53 acts as a key tumor suppressor by regulating cell cycle arrest, DNA repair, and apoptosis, and its antitumor activity is regulated by the hetero- and homo-oligomerization of MDMX and MDM2 proteins. So far, some traditional methods have been utilized to study the oligomerization of MDMX and MDM2 in vitro, but they have not clarified some controversial issues or whether the extracellular results can represent the intracellular results. Here, we put forward an in situ method for studying protein homo- and hetero-oligomerization in single living cells by using fluorescence correlation spectroscopy. In this study, MDMX and MDM2 were labeled with fluorescent proteins using lentiviral transfection. Autocorrelation spectroscopy and cross-correlation spectroscopy methods were used to study the oligomerization of MDMX and MDM2 in situ and the effect of regulation of MDMX oligomerization on p53-MDMX interactions in single living cells. We observed the homo- and hetero-oligomerization of MDMX and MDM2 in living cells. Meanwhile, the levels of the homo-oligomers of MDMX and MDM2 were increased due to the lack of hetero-oligomerization. Finally, the binding affinity of MDMX for p53 was improved with an increase in the level of MDMX hetero-oligomerization.
- Subjects :
- Cell cycle checkpoint
MDMX
biology
Chemistry
DNA repair
Nuclear Proteins
Apoptosis
Cell Cycle Proteins
Proto-Oncogene Proteins c-mdm2
Fluorescence correlation spectroscopy
Transfection
Biochemistry
Fluorescence
Spectrometry, Fluorescence
Proto-Oncogene Proteins
Biophysics
biology.protein
Humans
Protein oligomerization
Mdm2
Single-Cell Analysis
Tumor Suppressor Protein p53
In Situ Hybridization, Fluorescence
Intracellular
Protein Binding
Subjects
Details
- ISSN :
- 15204995 and 00062960
- Volume :
- 60
- Database :
- OpenAIRE
- Journal :
- Biochemistry
- Accession number :
- edsair.doi.dedup.....2a4ca35ab7e862042d1cee8e3150be49