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In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
Shanghai Jiao Tong Univ, Sch Life Sci & Biotechnol, State Key Lab Microbial Metab, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
Shanghai Jiao Tong Univ, Sch Chem & Chem Engn, State Key Lab Met Matrix Composites, 800 Dongchuan Rd, Shanghai 200240, Peoples R China..
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2018 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 90, no 10, p. 6144-6151Article in journal (Refereed) Published
Abstract [en]

Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53 MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of pS3-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3 alpha and MI773 efficiently inhibited the pS3-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein protein interactions and evaluation of inhibitors in living cells.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018. Vol. 90, no 10, p. 6144-6151
National Category
Chemical Sciences
Identifiers
URN: urn:nbn:se:kth:diva-229014DOI: 10.1021/acs.analchem.8b00473ISI: 000432478600026PubMedID: 29671327Scopus ID: 2-s2.0-85046367106OAI: oai:DiVA.org:kth-229014DiVA, id: diva2:1211601
Note

QC 20180531

Available from: 2018-05-31 Created: 2018-05-31 Last updated: 2019-04-04Bibliographically approved
In thesis
1. Super resolution fluorescence imaging: analyses, simulations and applications
Open this publication in new window or tab >>Super resolution fluorescence imaging: analyses, simulations and applications
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Fluorescence methods offer extraordinary sensitivity and specificity, and are extensively used in the life sciences. In recent years, super resolution fluorescence imaging techniques have developed strongly, uniquely combining ~10 nm sub diffraction resolution and specific labeling with high efficiency. This thesis explores this potential, with a major focus on Stimulated Emission Depletion, STED, microscopy, applications thereof, image analyses and simulation studies. An additional theme in this thesis is development and use of single molecule fluorescence correlation spectroscopy, FCS, and related techniques, as tools to study dynamic processes at the molecular level. In paper I the proteins cytochrome-bo3 and ATP-synthase are studied with fluorescence cross-correlation spectroscopy, FCCS. These two proteins are a part of the energy conversion process in E. coli, converting ADP into ATP. We found that an increased interaction between these proteins, detected by FCCS, correlates with an increase in the ATP production. In paper II an FCS-based imaging method is developed, capable to determine absolute sizes of objects, smaller than the resolution limit of the microscope used. Combined with STED, this may open for studies of membrane nano-domains, such as those investigated by simulations in paper VII. In paper III and paper IV super resolution STED imaging was applied on Streptococcus Pneumoniae, revealing information about function and distribution of proteins involved in the defense mechanism of the bacteria, as well as their role in bacterial meningitis. In paper V, we used STED imaging to investigate protein distributions in platelets. We then found that the adhesion protein P-selectin changes its distribution pattern in platelets incubated with tumor cells, and with machine learning algorithms and classical image analysis of the STED images it is possible to automatically distinguish such platelets from platelets activated by other means. This could provide a strategy for minimally invasive diagnostics of early cancer development, and deeper understanding of the role of platelets in cancer development. Finally, this thesis presents Monte-Carlo simulations of biological processes and their monitoring by FCS. In paper VI, a combination of FCCS and simulations was applied to resolve the interactions between a transcription factor (p53) and an oncoprotein (MDM2) inside live cells. In paper VII, the feasibility of FCS techniques for studying nano-domains in membranes is investigated purely by simulations, identifying the conditions under which such nano-domains would be possible to detect by FCS. In paper VIII, proton exchange dynamics at biological membranes were simulated in a model, verifying experimental FCS data and identifying fundamental mechanisms by which membranes mediate proton exchange on a local (~10nm) scale.

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. 81
Series
TRITA-SCI-FOU ; 2019:20
National Category
Other Physics Topics
Research subject
Physics
Identifiers
urn:nbn:se:kth:diva-248297 (URN)978-91-7873-171-8 (ISBN)
Public defence
2019-04-26, FA32, KTH, Roslagstullsbacken 21, Stockholm, 18:22 (English)
Opponent
Supervisors
Note

QC 20190405

Available from: 2019-04-05 Created: 2019-04-04 Last updated: 2019-04-05Bibliographically approved

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